U.S. patent application number 15/107502 was filed with the patent office on 2017-06-29 for method for diagnosis of alzheimer's disease and frontotemporal lobar degeneration, diagnostic agent, therapeutic agent, and screening method for said agents.
This patent application is currently assigned to National University Corporation Tokyo Medical and Dental University. The applicant listed for this patent is NATIONAL UNIVERSITY CORPORATION TOKYO MEDICAL AND DENTAL UNIVERSITY. Invention is credited to Hitoshi OKAZAWA.
Application Number | 20170182012 15/107502 |
Document ID | / |
Family ID | 53478934 |
Filed Date | 2017-06-29 |
United States Patent
Application |
20170182012 |
Kind Code |
A1 |
OKAZAWA; Hitoshi |
June 29, 2017 |
METHOD FOR DIAGNOSIS OF ALZHEIMER'S DISEASE AND FRONTOTEMPORAL
LOBAR DEGENERATION, DIAGNOSTIC AGENT, THERAPEUTIC AGENT, AND
SCREENING METHOD FOR SAID AGENTS
Abstract
It has been revealed that, from a pre-onset stage of Alzheimer's
disease, enhancement of phosphorylations of MARCKS and the like
causes abnormal spine formation or the like, consequently
developing the disease. Moreover, it has also been revealed that
the phosphorylations of MARCKS and the like are caused by PKC and
the like, and further that b-raf is involved in the phosphorylation
of a tau protein important for the progression of Alzheimer's
disease. Thus, these proteins have been found to be target
molecules useful in the diagnosis and treatment of Alzheimer's
disease. In addition, it has also been revealed that, in a
pre-onset stage of frontotemporal lobar degeneration also, b-RAF
phosphorylation enhancement causes a decrease in the number of
spines and the like, consequently developing the disease. Thus,
b-RAF has been found to be a target molecule useful in the
diagnosis and treatment of frontotemporal lobar degeneration.
Inventors: |
OKAZAWA; Hitoshi; (Tokyo,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NATIONAL UNIVERSITY CORPORATION TOKYO MEDICAL AND DENTAL
UNIVERSITY |
Tokyo |
|
JP |
|
|
Assignee: |
National University Corporation
Tokyo Medical and Dental University
Tokyo
JP
|
Family ID: |
53478934 |
Appl. No.: |
15/107502 |
Filed: |
December 25, 2014 |
PCT Filed: |
December 25, 2014 |
PCT NO: |
PCT/JP2014/084424 |
371 Date: |
September 23, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2800/2821 20130101;
A61P 25/28 20180101; A61K 31/506 20130101; G01N 33/6896 20130101;
A61P 43/00 20180101; G01N 2500/04 20130101; A61K 31/44 20130101;
A61K 31/437 20130101; A61K 31/4439 20130101; G01N 2500/20 20130101;
C12Q 1/485 20130101 |
International
Class: |
A61K 31/437 20060101
A61K031/437; G01N 33/68 20060101 G01N033/68; C12Q 1/48 20060101
C12Q001/48 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 27, 2013 |
JP |
2013-272189 |
Claims
1. A method for diagnosing Alzheimer's disease, the method
comprising: (i) a step of detecting, in a test subject, a
phosphorylation of at least one substrate protein selected from the
group consisting of MARCKS, Marcksl1, SRRM2, SPTA2, ADDB, NEUM,
BASP1, SYT1, G3P, HS90A, CLH, NFH, NFL, GPRIN1, ACON, ATPA, and
ATPB; (ii) a step of comparing the phosphorylation with a
phosphorylation of a substrate protein in a normal subject; and
(iii) a step of determining that the test subject is affected with
Alzheimer's disease or has a risk of developing Alzheimer's disease
if the phosphorylation of the substrate protein in the test subject
is higher than the phosphorylation of the substrate protein in the
normal subject as a result of the comparison.
2. A method for diagnosing Alzheimer's disease, the method
comprising: (i) a step of detecting an activity or expression of a
kinase protein in a test subject; (ii) a step of comparing the
activity or expression with an activity or expression of a kinase
protein in a normal subject; and (iii) a step of determining that
the test subject is affected with Alzheimer's disease or has a risk
of developing Alzheimer's disease if the activity or expression of
the kinase protein in the test subject is higher than the activity
or expression of the kinase protein in the normal subject as a
result of the comparison, wherein the kinase protein is at least
one kinase protein selected from the group consisting of PKC, CaMK,
CSK, Lyn, and b-RAF.
3. An agent for diagnosing Alzheimer's disease, the agent
comprising a compound having an activity of binding to a
phosphorylation site of at least one substrate protein selected
from the group consisting of MARCKS, Marcksl1, SRRM2, SPTA2, ADDB,
NEUM, BASP1, SYT1, G3P, HS90A, CLH, NFH, NFL, GPRIN1, ACON, ATPA,
and ATPB.
4. An agent for diagnosing Alzheimer's disease, the agent
comprising a compound having an activity of binding to at least one
kinase protein selected from the group consisting of PKC, CaMK,
CSK, Lyn, and b-RAF.
5. A screening method for a candidate compound for diagnosing
Alzheimer's disease, the method comprising the steps of: bringing a
test compound into contact with a phosphorylation site of at least
one substrate protein selected from the group consisting of MARCKS,
Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A, CLH,
NFH, NFL, GPRIN1, ACON, ATPA, and ATPB; and selecting the compound
if the compound binds to the phosphorylation site.
6. A screening method for a candidate compound for diagnosing
Alzheimer's disease, the method comprising the steps of: bringing a
test compound into contact with at least one kinase protein
selected from the group consisting of PKC, CaMK, CSK, Lyn, and
b-RAF; and selecting the compound if the compound binds to the
kinase protein.
7. An agent for treating Alzheimer's disease, the agent comprising
a compound capable of suppressing a phosphorylation of at least one
substrate protein selected from the group consisting of MARCKS,
Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A, CLH,
NFH, NFL, GPRIN1, ACON, ATPA, and ATPB.
8. An agent for treating Alzheimer's disease, the agent comprising
a compound capable of suppressing an activity or expression of at
least one kinase protein selected from the group consisting of PKC,
CaMK, CSK, Lyn, and b-RAF.
9. The agent according to claim 8, wherein the compound is capable
of suppressing an activity or expression of b-RAF and is at least
one compound selected from the group consisting of PLX-4720,
sorafenib, GDC-0879, vemurafenib, dabrafenib, sorafenib tosylate,
and LGX818.
10. The agent according to claim 9, wherein the compound is
vemurafenib.
11. An agent for treating Alzheimer's disease, the agent comprising
a compound capable of suppressing a binding of at least one
substrate protein selected from the group consisting of MARCKS,
Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A, CLH,
NFH, NFL, GPRIN1, ACON, ATPA, and ATPB to at least one kinase
protein selected from the group consisting of PKC, CaMK, CSK, Lyn,
and b-RAF.
12. A screening method for a candidate compound for treating
Alzheimer's disease, the method comprising: (i) a step of applying
a test compound to a system capable of detecting a phosphorylation
of at least one substrate protein selected from the group
consisting of MARCKS, Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1,
SYT1, G3P, HS90A, CLH, NFH, NFL, GPRIN1, ACON, ATPA, and ATPB; and
(ii) a step of selecting the compound if the compound suppresses
the phosphorylation of the substrate protein.
13. A screening method for a candidate compound for treating
Alzheimer's disease, the method comprising: (i) a step of applying
a test compound to a system capable of detecting an activity or
expression of at least one kinase protein selected from the group
consisting of PKC, CaMK, CSK, Lyn, and b-RAF; and (ii) a step of
selecting the compound if the compound suppresses the activity or
expression of the protein.
14. A screening method for a candidate compound for treating
Alzheimer's disease, the method comprising the following steps (a)
to (c): (a) a step of bringing at least one kinase protein selected
from the group consisting of PKC, CaMK, CSK, Lyn, and b-RAF into
contact with at least one substrate protein selected from the group
consisting of MARCKS, Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1,
SYT1, G3P, HS90A, CLH, NFH, NFL, GPRIN1, ACON, ATPA, and ATPB, in
presence of a test compound; (b) a step of detecting a binding
between the kinase protein and the substrate protein; and (c) a
step of selecting the compound if the compound suppresses the
binding.
15. An agent for treating frontotemporal lobar degeneration, the
agent comprising a compound capable of suppressing an activity or
expression of b-RAF.
16. The agent according to claim 15, wherein the compound is at
least one compound selected from the group consisting of PLX-4720,
sorafenib, GDC-0879, vemurafenib, dabrafenib, sorafenib tosylate,
and LGX818.
17. The agent according to claim 16, wherein the compound is
vemurafenib.
18. An agent for diagnosing frontotemporal lobar degeneration, the
agent comprising a compound having an activity of binding to b-RAF.
Description
TECHNICAL FIELD
[0001] The present invention relates to a diagnosis method, a
diagnostic agent, and a therapeutic agent against Alzheimer's
disease. Further, the present invention relates to a screening
method for candidate compounds of these agents. Moreover, the
present invention relates to a diagnostic agent and a therapeutic
agent against frontotemporal lobar degeneration.
BACKGROUND ART
[0002] Alzheimer's disease (Alzheimer's dementia, AD) is a
progressive neurodegenerative disease that may occur in presenile
to senile stages. The main symptoms include memory disorder, higher
brain function disorders (aphasia, apraxia, agnosia, constructional
apraxia), change in personality, and so forth. In addition, because
of such symptoms, the disease not only reduces the quality of life
of a patient himself/herself, but also greatly influences the
living styles of family and so on around the patient. Further, the
number of the patients is steadily increasing along with population
ageing. Alzheimer's disease is a serious problem of modern society
all over the world. Hence, Alzheimer's disease has been studied
actively, but the elucidation of the full onset mechanism thereof
and the development of an eradicative medicine have not been
achieved yet under current situations.
[0003] On the other hand, it is now possible to delay the
progression of Alzheimer's disease symptoms more than ever.
Particularly, cholinesterase inhibitors have been actually used in
clinical settings, resulting in some reasonable outcomes. The
progression of the symptoms can also be suppressed to some degree
currently. As a result, there is a demand in the treatment of
Alzheimer's disease that the disease should be detected at earlier
stages, there by hastening developments of: electroencephalography,
biochemical tests targeting blood and cerebrospinal fluid,
diagnostic imagings such as CT, MRI, and PET/SPECT, and so forth.
Particularly, PET is about to enable the detection of senile plaque
(amyloid plaque) deposition in the brain, which is a characteristic
of Alzheimer's disease and the most likely causative factor
thereof. However, currently-available diagnostic techniques still
have difficulty grasping a pre-onset stage of Alzheimer's disease
is developed, and no effective early-stage diagnosis method has
been established yet under current situations.
[0004] Besides senile plaque deposition, Alzheimer's disease is
neuropathologically characterized also by neurofibrillary tangle
(paired helical filament (PHF)) deposition. In addition, the
deposition of these structures is believed to cause nerve function
disorder and nerve cell death (nerve cell dropout) involved in the
symptoms described above. Moreover, it has been revealed that
senile plaques are structures formed when polypeptides, each
composed of approximately 40 amino acids, called amyloid .beta.
(A.beta.) aggregate and deposit outside nerve cells in high
density. Further, neurofibrillary tangles have been revealed to be
also structures formed when microtubule-associated proteins tau are
phosphorylated and thereby dissociated from cytoskeleton-forming
microtubules, followed by polymerization among the tau proteins.
Meanwhile, although no conclusion has been drawn yet regarding the
Alzheimer causative factor and onset mechanism, the most likely
mechanism is such that when amyloid .beta. molecules aggregate
(amyloid pathology), the aggregation promotes the tau
phosphorylation and polymerization (tau pathology), consequently
leading to nerve cell death and so forth (amyloid cascade
hypothesis).
[0005] Furthermore, it is suggested that various phosphorylation
signal transductions are involved in a pathology of Alzheimer's
disease. For example, as described above, the deposition of
neurofibrillary tangles is due to tau phosphorylation. It is also
revealed that this tau phosphorylation is regulated by various
serine/threonine kinases such as GSK3.beta., JNK, PKA, Cdk5, and
casein kinase II (NPLs 1 to 7). Moreover, it has been presumed that
microtubules from which tau is dissociated by such phosphorylation
become unstable, consequently decreasing neurites as observed in
the brains of AD patients (NPLs 8 and 9).
[0006] In addition, it is suggested that a phosphorylation enzyme
PKC is involved in memory formation (NPLs 10 and 11). Further,
activating PKC and CaMKII is believed to promote the transcriptions
of BDNF and Arc involved in memory control, and also have a
protective function against Alzheimer's disease (NPLs 12 and 13).
Additionally, based on such findings, an attempt has been made to
apply PKC activators such as bryostatin in the treatment of
Alzheimer's disease. However, it is also reported that an excessive
activation of PKC, on the other hand, impairs working memory (NPL
14).
[0007] Furthermore, it is also suggested that the phosphorylation
of MARCKS by PKC dissociates this protein from the cell membrane
(PIP2 and actins) and, as a result, induces amyloid .beta.
production (NPL 15). Moreover, phosphorylated MARCKS is observed in
dystrophic neurites and microglia within senile plaques.
Nevertheless, it has also been revealed that the phosphorylation
level of MARCKS in the brains of Alzheimer's disease patients is
lower than that of healthy subjects (NPL 16).
[0008] As described above, it has been suggested that
phosphorylation signal transductions are involved in a pathology of
Alzheimer's disease. If this involvement can be elucidated in more
details, it is expected to greatly contribute to establishments of
early-stage diagnosis and treatment methods against this
disease.
[0009] Nonetheless, in a phosphorylation signal transduction,
particularly, in a wide variety of phosphorylation signal
transductions in Alzheimer's disease, what protein phosphorylations
play a central role in a pre-onset stage of Alzheimer's disease has
not been elucidated at all yet.
[0010] Meanwhile, frontotemporal lobar degeneration (FTLD) is known
as a disease that exhibits progressive neurodegenerative disorders
like Alzheimer's disease. Frontotemporal lobar degeneration is the
second or third most frequent early-onset neurodegenerative
dementia after Alzheimer's disease. The symptoms to be exhibited
include drastic changes in behavior and personality. A language
function disorder occurs together with FTLD in many cases, and
gradually develops into a cognitive disorder and dementia. In
addition, the studies have been conducted as in the case of
Alzheimer's disease, but the full onset mechanism of FTLD has not
been revealed yet.
[0011] For example, it is known that one cause of genetic
frontotemporal lobar degeneration is a mutation in the PGRN gene.
Moreover, there is a report that the PGRN protein exhibits an
antagonistic action against TNF in binding to TNF receptors,
suggesting that this antagonism is involved in the onset of
frontotemporal lobar degeneration (NPL 17). However, on the other
hand, contradictory results are also reported. The molecular
mechanism in the onset of frontotemporal lobar degeneration,
including the possibility of the involvement of the TNF signal
transduction pathway (NPLs 18 to 21), has not been elucidated under
current situations.
[0012] Additionally, for the elucidation of the molecular mechanism
of frontotemporal lobar degeneration, PGRN gene knockout mice have
been prepared as model animals. Moreover, findings (such as
excessive inflammatory reaction, cellular ageing, synaptic
dysfunction, ubiquitination promotion, increased caspase
activation, decreased TDP-43 solubility) exhibited in
frontotemporal lobar degeneration are actually observed in such
knockout mice (NPLs 22 to 28). However, as has been pointed out in
other neurodegenerative diseases such as Alzheimer's disease also
(NPL 29), artificially reducing the amount of the PGRN protein in
the model animals actually results in mere mimicking of symptoms
caused by the expressions of mutated PGRN mRNA and the like, so
that the effectiveness as model animals is questionable.
[0013] As has been described above, no effective model animal is
developed against frontotemporal lobar degeneration, and the onset
mechanism has not been elucidated as in the case of Alzheimer's
disease. Hence, in the development of diagnosis and treatment
methods against the disease, no useful target molecule has been
found under current situations.
CITATION LIST
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2014, Vol. 17, pp. 661 to 663
SUMMARY OF INVENTION
Technical Problem
[0043] The present invention has been made in view of the problems
of the above-described conventional techniques. An object of the
present invention is to identify phosphoproteins and kinase
proteins which play central roles in a pre-onset stage of
Alzheimer's disease, as well as a network composed of these
proteins, and consequently to provide target molecules useful in
the diagnosis and treatment of Alzheimer's disease.
[0044] In addition, another object of the present invention is to
identify a signal transduction pathway which plays a central role
in a pre-onset stage of frontotemporal lobar degeneration, and
consequently to provide target molecules useful in the diagnosis
and treatment of frontotemporal lobar degeneration.
Solution to Problem
[0045] In order to provide target molecules useful in the diagnosis
and treatment of Alzheimer's disease, the present inventor first
employed an analysis according to a mass spectrometry method (2D LC
MS/MS analysis) targeting brains at the pre-onset stage of tau
model mice and four types of Alzheimer's disease (AD) model mice
and on postmortem brains of AD patients, and searched 1100 or more
phosphoproteins and 30000 or more phosphopolypeptides for proteins
whose expression amounts changed in comparison with the respective
wild-type mice and healthy subjects, for example.
[0046] As a result, phosphoproteins whose expressions changed
immediately before or immediately after amyloid .beta. started
aggregating were successfully identified in the brains of multiple
AD model mice. Further, it was revealed that the phosphorylations
of most of these phosphoproteins also changed commonly in the AD
patients or the tau model mice. In sum, MARCKS, Marcksl1, SRRM2,
SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A, CLH, NFH, NFL, GPRIN1,
ACON, ATPA, and ATPB were successfully selected as proteins (AD
core proteins) whose phosphorylation levels changed in brains
affected with Alzheimer's disease, and which were presumed to play
central roles in the pathology.
[0047] Further, these AD core proteins were incorporated into the
experimentally verified protein-protein interaction (PPI) database
for the analysis. Thus, an AD signaling network (AD core network)
was identified which would serve as the core in the pathology of
Alzheimer's disease.
[0048] The result surprisingly revealed that most of the AD core
proteins directly interacted with each other, and further that
their functions were focused on important functions in synapse such
as spine formation, vesicle recycling, and energy metabolism.
Particularly, it was revealed that the enhancement of the
phosphorylations of the AD core proteins involved in nerve cell
skeleton started from a non-symptomatic stage before amyloid .beta.
aggregation. Further, it was possible to categorize the changes in
the AD core protein phosphorylations in a pre-onset stage of
Alzheimer's disease into three patterns: one having a peak at an
initial phase, one having a peak at a mid phase, and one having a
peak at a late phase. In this manner, it was also revealed that the
phosphorylation changed in each AD core protein in a time-specific
manner.
[0049] Further, the analysis utilizing the protein-protein
interaction database also enables the identifications of PKC, CaMK,
CSK, and Lyn as kinases which controlled such time-specific changes
in phosphorylations.
[0050] Meanwhile, it has been presumed that the transition from
amyloid .beta. aggregation (amyloid pathology) to tau
phosphorylation and polymerization (tau pathology) by the
aggregation plays an important role in the pathology of Alzheimer's
disease. In this regard, the result of the mass spectrometry on the
model mice also enabled the identification of b-RAF as a kinase
which promoted the transition from amyloid pathology to tau
pathology.
[0051] Furthermore, it was also verified that suppressing the
expression of MARCKS or suppressing the kinase activity of PKC or
CaMK enabled a recovery of Alzheimer's disease pathology (abnormal
spine formation). These have led to the completion of the present
invention.
[0052] Additionally, in order to provide target molecules useful in
the diagnosis and treatment of frontotemporal lobar degeneration
(FTLD), the present inventor first made efforts to prepare an
animal which could be said as a true FTLD model in view of the
current situation where the expressions of mutated mRNA and the
like, which would cause the disease, had not been reproduced by the
existing FTLD model animals as described above. To be more
specific, efforts were made to prepare FTLD model mice by
introducing a stop mutation, which was obsersed in FTLD patients,
into the PGRN (progranulin) gene of mice. As a result, it was found
out not only that the expressions of the mutated PGRN mRNA and a
mutant protein encoded thereby were observed in the obtained
PGRN-KI mice, but also that the introduction of the mutation
enabled reproduction of both the pathological observations and the
clinical symptoms of FTLD patients in the mice. Accordingly, it was
revealed that the PGRN-KI mice were quite useful as FTLD model
animals.
[0053] Next, using the PGRN-KI mice, efforts were made, as in the
case of the above Alzheimer's disease analysis, to comprehensively
analyze (phosphoproteome analysis) phosphorylation signal
transductions in FTLD also to identify a phosphorylation signal
transduction which played a central role in a pathology of the
disease.
[0054] As a result, surprisingly, it was found that no protein had
a change in phosphorylation in a TNF signal transduction pathway
per se which had been heretofore suggested to be involved in the
onset mechanism of FTLD. On the other hand, it was revealed that,
in TNF-related signal transduction pathways such as a MAPK signal
transduction pathway in the PGRN-KI mice, the phosphorylations of
proteins belonging to such signal transduction pathways were
remarkably changed. Particularly, a MAPK signal transduction
pathway was apparently activated in the PGRN-KI mice from the
pre-onset stage. During the period of symptom progression also,
multiple proteins belonging to the signal transduction pathway were
in high phosphorylation states all the time.
[0055] Hence, next, an analysis was performed for the therapeutic
effect of targeting b-RAF, its phosphorylation substrate tau, and
the like, which belonged to the MAPK signal transduction pathway,
and which were revealed to be in high phosphorylation states in the
PGRN-KI mice by the aforementioned analysis. To be more specific,
first, analyzed was whether or not suppressing an abnormal
activation in the MAPK signal transduction pathway by using a b-raf
specific inhibitor or the like would recover the phenotype of the
PGRN-KI mice.
[0056] The result revealed that administering the b-raf inhibitor
alleviated the abnormal behavior observed in the PGRN-KI mice.
Further, it was also revealed that administering the b-raf
inhibitor or knocking down tau recovered the number of spines which
was decreased in the PGRN-KI mice. These have led to the completion
of the present invention.
[0057] To be more specific, the present invention relates to a
diagnosis method, a diagnostic agent, a screening method for a
candidate compound of the diagnostic agent, a therapeutic agent,
and a screening method for a candidate compound of the therapeutic
agent all of which are against Alzheimer's disease and target the
above-described AD core proteins and kinases for phosphorylating
the proteins.
[0058] More specifically, the present invention provides the
following.
<1> A method for diagnosing Alzheimer's disease, the method
comprising:
[0059] (i) a step of detecting, in a test subject, a
phosphorylation of at least one substrate protein selected from the
group consisting of MARCKS, Marcksl1, SRRM2, SPTA2, ADDB, NEUM,
BASP1, SYT1, G3P, HS90A, CLH, NFH, NFL, GPRIN1, ACON, ATPA, and
ATPB;
[0060] (ii) a step of comparing the phosphorylation with a
phosphorylation of a substrate protein in a normal subject; and
[0061] (iii) a step of determining that the test subject is
affected with Alzheimer's disease or has a risk of developing
Alzheimer's disease if the phosphorylation of the substrate protein
in the test subject is higher than the phosphorylation of the
substrate protein in the normal subject as a result of the
comparison.
<2> A method for diagnosing Alzheimer's disease, the method
comprising:
[0062] (i) a step of detecting an activity or expression of a
kinase protein in a test subject;
[0063] (ii) a step of comparing the activity or expression with an
activity or expression of a kinase protein in a normal subject;
and
[0064] (iii) a step of determining that the test subject is
affected with Alzheimer's disease or has a risk of developing
Alzheimer's disease if the activity or expression of the kinase
protein in the test subject is higher than the activity or
expression of the kinase protein in the normal subject as a result
of the comparison, wherein [0065] the kinase protein is at least
one kinase protein selected from the group consisting of PKC, CaMK,
CSK, Lyn, and b-RAF. <3> An agent for diagnosing Alzheimer's
disease, the agent comprising a compound having an activity of
binding to a phosphorylation site of at least one substrate protein
selected from the group consisting of MARCKS, Marcksl1, SRRM2,
SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A, CLH, NFH, NFL, GPRIN1,
ACON, ATPA, and ATPB. <4> An agent for diagnosing Alzheimer's
disease, the agent comprising a compound having an activity of
binding to at least one kinase protein selected from the group
consisting of PKC, CaMK, CSK, Lyn, and b-RAF. <5> A screening
method for a candidate compound for diagnosing Alzheimer's disease,
the method comprising the steps of:
[0066] bringing a test compound into contact with a phosphorylation
site of at least one substrate protein selected from the group
consisting of MARCKS, Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1,
SYT1, G3P, HS90A, CLH, NFH, NFL, GPRIN1, ACON, ATPA, and ATPB;
and
[0067] selecting the compound if the compound binds to the
phosphorylation site.
<6> A screening method for a candidate compound for
diagnosing Alzheimer's disease, the method comprising the steps
of:
[0068] bringing a test compound into contact with at least one
kinase protein selected from the group consisting of PKC, CaMK,
CSK, Lyn, and b-RAF; and
[0069] selecting the compound if the compound binds to the kinase
protein.
<7> An agent for treating Alzheimer's disease, the agent
comprising a compound capable of suppressing a phosphorylation of
at least one substrate protein selected from the group consisting
of MARCKS, Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P,
HS90A, CLH, NFH, NFL, GPRIN1, ACON, ATPA, and ATPB. <8> An
agent for treating Alzheimer's disease, the agent comprising a
compound capable of suppressing an activity or expression of at
least one kinase protein selected from the group consisting of PKC,
CaMK, CSK, Lyn, and b-RAF. <9> The agent according to
<8>, wherein the compound is capable of suppressing an
activity or expression of b-RAF and is at least one compound
selected from the group consisting of PLX-4720, sorafenib,
GDC-0879, vemurafenib, dabrafenib, sorafenib tosylate, and LGX818.
<10> The agent according to <9>, wherein the compound
is vemurafenib. <11> An agent for treating Alzheimer's
disease, the agent comprising a compound capable of suppressing a
binding of at least one substrate protein selected from the group
consisting of MARCKS, Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1,
SYT1, G3P, HS90A, CLH, NFH, NFL, GPRIN1, ACON, ATPA, and ATPB to at
least one kinase protein selected from the group consisting of PKC,
CaMK, CSK, Lyn, and b-RAF. <12> A screening method for a
candidate compound for treating Alzheimer's disease, the method
comprising: [0070] (i) a step of applying a test compound to a
system capable of detecting a phosphorylation of at least one
substrate protein selected from the group consisting of MARCKS,
Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A, CLH,
NFH, NFL, GPRIN1, ACON, ATPA, and ATPB; and [0071] (ii) a step of
selecting the compound if the compound suppresses the
phosphorylation of the substrate protein. <13> A screening
method for a candidate compound for treating Alzheimer's disease,
the method comprising: [0072] (i) a step of applying a test
compound to a system capable of detecting an activity or expression
of at least one kinase protein selected from the group consisting
of PKC, CaMK, CSK, Lyn, and b-RAF; and [0073] (ii) a step of
selecting the compound if the compound suppresses the activity or
expression of the protein. <14> A screening method for a
candidate compound for treating Alzheimer's disease, the method
comprising the following steps (a) to (c): [0074] (a) a step of
bringing at least one kinase protein selected from the group
consisting of PKC, CaMK, CSK, Lyn, and b-RAF into contact with at
least one substrate protein selected from the group consisting of
MARCKS, Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P,
HS90A, CLH, NFH, NFL, GPRIN1, ACON, ATPA, and ATPB, in presence of
a test compound; [0075] (b) a step of detecting a binding between
the kinase protein and the substrate protein; and [0076] (c) a step
of selecting the compound if the compound suppresses the
binding.
[0077] In addition, the present invention relates to a therapeutic
agent and a diagnostic agent which are against frontotemporal lobar
degeneration and target b-RAF described above. More specifically,
the present invention provides the following.
<15> An agent for treating frontotemporal lobar degeneration,
the agent comprising a compound capable of suppressing an activity
or expression of b-RAF. <16> The agent according to
<15>, wherein the compound is at least one compound selected
from the group consisting of PLX-4720, sorafenib, GDC-0879,
vemurafenib, dabrafenib, sorafenib tosylate, and LGX818. <17>
The agent according to <16>, wherein the compound is
vemurafenib. <18> An agent for diagnosing frontotemporal
lobar degeneration, the agent comprising a compound having an
activity of binding to b-RAF.
[0078] It should be noted that, in the present invention,
"Alzheimer's disease" is a neurodegenerative disease also referred
to as Alzheimer's dementia or AD, and includes "familial
Alzheimer's disease" and "inherited Alzheimer's disease"
attributable to gene mutation, and also "sporadic Alzheimer's
disease" due to environmental factors such as lifestyle and stress.
"Developing Alzheimer's disease" and related phrases mean an
expression of symptoms such as memory disorder, higher brain
function disorders (aphasia, apraxia, agnosia, constructional
apraxia), and change in personality judged by clinical diagnosis,
as well as appearance of atrophy in the brain judged by diagnostic
imaging. "Affected with Alzheimer's disease" and related phrases
mean to also include a state where the symptoms are not expressed,
but a pathological change peculiar to Alzheimer's disease (for
example, amyloid .beta. aggregation) occurs.
[0079] In addition, in the present invention, "frontotemporal lobar
degeneration" is a non-Alzheimer's disease type neurodegenerative
disease also referred to as FTLD, by which atrophy occurs in the
frontal lobe and temporal lobe at the early stage, and atrophy
occurs throughout the brain at the late stage. To be more specific,
frontotemporal lobar degeneration includes three diseases
classified according to clinical characteristics: frontotemporal
dementia (FTD), progressive nonfluent aphasia (PNFA), and semantic
dementia (SD). Moreover, frontotemporal lobar degeneration includes
four diseases pathologically classified into FTLD-Tau, FTLD-TDP,
FTLD-UPS, and FTLD-FUS according to the type of proteins
accumulated as abnormal proteins in cells.
[0080] Further, "FTLD-Tau" is classified into "3R Tau" type, "4R
Tau" type, and "3/4R Tau" type according to the number of
microtubule-binding regions repeated in tau proteins predominantly
accumulated in cells. Moreover, the "3R Tau" type includes FTLD
with Pick bodies (Pick's disease), FTLD with MAPT
(microtubule-associated protein tau) gene mutation (FTLD-17), and
the like. The "4R Tau" type includes corticobasal degeneration,
progressive supranuclear palsy, multiple system tauopathy with
dementia, argyrophilic grain dementia (argyrophilic grain disease),
FTLD with MAPT gene mutation (FTLD-17), and the like. The "3/4R
Tau" type includes dementia with neurofibrillary tangles, FTLD with
MAPT gene mutation (FTLD-17), and the like. On the other hand, a
FTLD group having tau-negative, ubiquitin-positive inclusions is
called "FTLD-U", and includes FTLD-TDP, FTLD-UPS and FTLD-FUS
described above.
[0081] Among FTLD-U, "FTLD-TDP" means a TDP-43-positive disease.
This disease includes FTLD with PGRN (progranulin gene) mutation,
sporadic FTLD-TDP/FTLD-U, FTLD with TARDBP (TDP-43 gene) mutation,
FTLD with VCP (valosi-containing protein gene) mutation, FTLD
linked to chromosome 9, and the like. Moreover, "FTLD-FUS" among
FTLD-U means TDP-43-negative, FUS (fused in sarcoma)-positive
disease. This disease includes neuronal intermediate filament
inclusion disease, non-typical FTLD-U, basophilic inclusion body
disease, FTLD with FUS mutation, and the like.
[0082] Further, "FTLD-UPS" is one of TDP-43-negative FTLD-U, This
disease includes FTLD with CHMP2B (charged multivesicular body
protein 2B gene) mutation, and the like.
[0083] "Developing frontotemporal lobar degeneration" and related
phrases mean an expression of symptoms such as memory disorder,
higher brain function disorders (aphasia, apraxia, agnosia,
constructional apraxia), and change in personality judged by
clinical diagnosis, as well as appearance of atrophy in the brain
judged by diagnostic imaging. "Affected with frontotemporal lobar
degeneration" and related phrases mean to also include a state
where the symptoms are not expressed, but a pathological change
peculiar to frontotemporal lobar degeneration (for example,
accumulation of the abnormal proteins in cells) occurs.
[0084] The "test subject" is a subject of the diagnosis method of
the present invention, and includes not only bodies of animals
including human, but also body fluids, tissues, cells, and the like
(for example, cerebrospinal fluid, cranial nerve tissues
(particularly neurological bioptic tissues), blood, blood plasma,
serous fluid, lymph, urine, saliva) isolated from the bodies.
[0085] The term "normal" in the normal subject means a state where
the subject is not affected with at least the disease (Alzheimer's
disease or frontotemporal lobar degeneration) to be targeted by the
diagnosis method of the present invention. Additionally, in the
diagnosis method of the present invention, a normal subject used as
a comparison target of a test subject is preferably the same gender
as the test subject and similar in age.
[0086] Furthermore, in the present invention, "MARCKS" is a protein
also referred to as myristoylated alanine-rich C-kinase substrate.
A typical human-derived example thereof includes a protein
specified under RefSeq ID: NP_002347. Moreover, a typical example
of a human-derived nucleic acid encoding MARCKS includes a nucleic
acid containing a coding region (CDS) represented by RefSeq ID:
NM_002358.
[0087] "Marcksl1" is a protein also referred to as MARCKS-like
protein 1. A typical human-derived example thereof includes a
protein specified under RefSeq ID: NP_075385. Moreover, a typical
example of a human-derived nucleic acid encoding MARCKS includes a
nucleic acid containing a CDS represented by RefSeq ID:
NM_023009.
[0088] "SRRM2" is a protein also referred to as
SRm300/serine-arginine repetitive matrix protein 2. A typical
human-derived example thereof includes a protein specified under
RefSeq ID: NP_057417. Moreover, a typical example of a
human-derived nucleic acid encoding SRRM2 includes a nucleic acid
containing a CDS represented by RefSeq ID: NM_016333.
[0089] "SPTA2" is a protein also referred to as .alpha.-II
spectrin. Typical human-derived examples thereof include a protein
specified under RefSeq NP_001123910, a protein specified under
RefSeq NP_001182461, and a protein specified under RefSeq
NP_003118. Moreover, typical examples of a human-derived nucleic
acid encoding SPTA2 include a nucleic acid containing a CDS
represented by RefSeq NM_001130438, a nucleic acid containing a CDS
represented by RefSeq NM_001195532, and a nucleic acid containing a
CDS represented by RefSeq NM_003127.
[0090] "ADDB" is a protein also referred to as .beta. adducin.
Typical human-derived examples thereof include a protein specified
under RefSeq NP_001171983, a protein specified under
RefSeqNP_001171984, a protein specified under RefSeq NP_001608, a
protein specified under RefSeq NP_059516, and a protein specified
under RefSeq NP_059522. Moreover, typical examples of a
human-derived nucleic acid encoding ADDB include a nucleic acid
containing a CDS represented by RefSeq NM_001185054, a nucleic acid
containing a CDS represented by RefSeq NM_001185055, a nucleic acid
containing a CDS represented by RefSeqNM_001617, a nucleic acid
containing a CDS represented by RefSeq NM_017482, and a nucleic
acid containing a CDS represented by RefSeq NM_017488.
[0091] "NEUM" is a protein also referred to as neuromodulin or
GAP43. Typical human-derived examples thereof include a protein
specified under RefSeq NP_00112353 and a protein specified under
RefSeq NP_002036. Moreover, typical examples of a human-derived
nucleic acid encoding NEUM include a nucleic acid containing a CDS
represented by RefSeq NM_001130064 and a nucleic acid containing a
CDS represented by RefSeq NM_002045.
[0092] "BASP1" is a protein also referred to as NAP-22 or CAP23.
Typical human-derived examples thereof include a protein specified
under RefSeq NP_001258535 and a protein specified under RefSeq
NP_006308. Moreover, typical examples of a human-derived nucleic
acid encoding BASP1 include a nucleic acid containing a CDS
represented by RefSeq NM_001271606 and a nucleic acid containing a
CDS represented by RefSeq NM_006317.
[0093] "SYT1" is a protein also referred to as synaptotagmin 1.
Typical human-derived examples thereof include a protein specified
under RefSeq NP_00112927, a protein specified under RefSeq
NP_001129278, and a protein specified under RefSeq NP_005630.
Moreover, typical examples of a human-derived nucleic acid encoding
SYT1 include a nucleic acid containing a CDS represented by RefSeq
NM_001135805, a nucleic acid containing a CDS represented by RefSeq
NM_001135806, and a nucleic acid containing a CDS represented by
RefSeq NM_005639.
[0094] "G3P" is a protein also referred to as
glyceraldehyde-3-phosphate dehydrogenase. Typical human-derived
examples thereof include a protein specified under RefSeq
NP_001243728 and a protein specified under RefSeq NP_002037.
Moreover, typical examples of a human-derived nucleic acid encoding
G3P include a nucleic acid containing a CDS represented by RefSeq
NM_001256799 and a nucleic acid containing a CDS represented by
RefSeq NM 002046.
[0095] "HS90A" is a protein also referred to as HSP90,
HSP90.alpha., or HSP86. Typical human-derived examples thereof
include a protein specified under RefSeqNP_001017963 and a protein
specified under RefSeq NP_005339. Moreover, typical examples of a
human-derived nucleic acid encoding HS90A include a nucleic acid
containing a CDS represented by RefSeq NM_001017963 and a nucleic
acid containing a CDS represented by RefSeq NM_005348.
[0096] "CLH" is a protein also referred to as CLH1 or clathrin
heavy chain 1. A typical human-derived example thereof includes a
protein specified under NP_004850. Moreover, a typical example of a
human-derived nucleic acid encoding CLH includes a nucleic acid
containing a CDS represented by NM_004859.
[0097] "NFH" is a protein also referred to as neurofilament heavy
polypeptide. A typical human-derived example thereof includes a
protein specified under RefSeqNP_066554. Moreover, a typical
example of a human-derived nucleic acid encoding NFH includes a
nucleic acid containing a CDS represented by RefSeq NM_021076.
[0098] "NFL" is a protein also referred to as neurofilament light
polypeptide. A typical human-derived example thereof includes a
protein specified under RefSeqNP_006149. Moreover, a typical
example of a human-derived nucleic acid encoding NFL includes a
nucleic acid containing a CDS represented by RefSeq NM_006158.
[0099] "GPRIN1" is a protein also referred to as G protein
regulated inducer 1. Typical human-derived examples thereof include
a protein specified under RefSeq NP_443131.2 and a protein
specified under RefSeq XP_005265863. Moreover, typical examples of
a human-derived nucleic acid encoding GPRIN1 include a nucleic acid
containing a CDS represented by RefSeq NM_052899 and a nucleic acid
containing a CDS represented by RefSeq XM_005265806.
[0100] "ACON" is a protein also referred to as aconitate hydratase.
A typical human-derived example thereof includes a protein
specified under RefSeq NP_001089. Moreover, a typical example of a
human-derived nucleic acid encoding ACON includes a nucleic acid
containing a CDS represented by RefSeq NM_001098.
[0101] "ATPA" is a protein also referred to as ATP synthase subunit
.alpha., ATP5A1, ATP5A, ATP5AL2, or ATPM. Typical human-derived
examples thereof include a protein specified under
RefSeqNP_001001935, a protein specified under RefSeq NP_001001937,
a protein specified under RefSeq NP_001244263, a protein specified
under RefSeq NP_001244264, and a protein specified under RefSeq
NP_004037. Moreover, typical examples of a human-derived nucleic
acid encoding ATPA include a nucleic acid containing a CDS
represented by RefSeq NM_001001935, a nucleic acid containing a CDS
represented by RefSeq NM_001001937, a nucleic acid containing a CDS
represented by RefSeq NM_001257334, a nucleic acid containing a CDS
represented by RefSeq NM_001257335, and a nucleic acid containing a
CDS represented by RefSeq NM_004046.
[0102] "ATPB" is a protein also referred to as ATP synthase subunit
.alpha., ATPMB, or ATPSB. A typical human-derived example thereof
includes a protein specified under RefSeq NP_001677. Moreover, a
typical example of a human-derived nucleic acid encoding ATPB
includes a nucleic acid containing a CDS represented by RefSeq
NM_001686.
[0103] Further, in the present invention, "PKC" is a protein also
referred to as protein kinase C. Examples thereof include
PKC.beta., PKC.alpha., PKC.lamda./.left brkt-bot. (lambda/iota),
PKC.sigma. (delta), and PKC.xi. (zeta).
[0104] Typical examples of human-derived "PKC.beta." include a
protein specified under RefSeq NP_002729 and a protein specified
under RefSeq NP_997700. Moreover, a typical example of a
human-derived nucleic acid encoding PKC.beta. includes a nucleic
acid containing a CDS represented by RefSeq NM_002738 and a nucleic
acid containing a CDS represented by RefSeq NM_212535.
[0105] A typical example of human-derived "PKC.alpha." includes a
protein specified under RefSeq NP_002728. Moreover, a typical
example of a human-derived nucleic acid encoding PKC.alpha.
includes a nucleic acid containing a CDS represented by RefSeq
NM_002737.
[0106] A typical example of human-derived "PKC.lamda./.left
brkt-bot." includes a protein specified under RefSeq NP_002731.
Moreover, a typical example of a human-derived nucleic acid
encoding PKC.lamda./.left brkt-bot. includes a nucleic acid
containing a CDS represented by RefSeq NM_002740.
[0107] Typical examples of human-derived "PKC.sigma." include a
protein specified under RefSeq NP_006245 and a protein specified
under RefSeq NP_997704. Moreover, typical examples of a
human-derived nucleic acid encoding PKC.sigma. include a nucleic
acid containing a CDS represented by RefSeq NM_006254 and a nucleic
acid containing a CDS represented by RefSeq NM_212539.
[0108] Typical examples of human-derived "PKC.xi." include a
protein specified under RefSeq NP_001028753, a protein specified
under RefSeq NP_001028754, a protein specified under RefSeq
NP_001229803, and a protein specified under RefSeq NP_002735.
Moreover, typical examples of a human-derived nucleic acid encoding
PKC.xi. include a nucleic acid containing a CDS represented by
RefSeq NM_001033581, a nucleic acid containing a CDS represented by
RefSeq NM_001033582, a nucleic acid containing a CDS represented by
RefSeq NM_001242874, and a nucleic acid containing a CDS
represented by RefSeq NM_002744.
[0109] "CaMK" is a protein also referred to as calmodulin kinase or
calmodulin-dependent protein kinase. Examples thereof include
CaMKI, CaMKII.beta., CaMKIV, CaMKII.sigma. (delta), and
CaMKII.alpha..
[0110] A typical example of human-derived "CaMKI" includes a
protein specified under RefSeq NP_003647. Moreover, a typical
example of a human-derived nucleic acid encoding CaMKI includes a
nucleic acid containing a CDS represented by RefSeq NM_003656.
[0111] Typical human-derived examples of "CaMKII.beta." include a
protein specified under RefSeq NP_001211, a protein specified under
RefSeqNP_742075, a protein specified under RefSeq NP_742076, a
protein specified under RefSeq NP_742077, a protein specified under
RefSeq NP_742078, a protein specified under RefSeq NP_742079, a
protein specified under RefSeqNP_742080, a protein specified under
RefSeq NP_742081, and a protein specified under RefSeq
XP_005249918. Moreover, typical examples of a human-derived nucleic
acid encoding CaMKII.beta. include a nucleic acid containing a CDS
represented by RefSeq NM_001220, a nucleic acid containing a CDS
represented by RefSeq NM_172078, a nucleic acid containing a CDS
represented by RefSeqNM_172079, a nucleic acid containing a CDS
represented by RefSeq NM_172080, a nucleic acid containing a CDS
represented by RefSeqNM_172081, a nucleic acid containing a CDS
represented by RefSeq NM_172082, a nucleic acid containing a CDS
represented by RefSeq NM_172083, a nucleic acid containing a CDS
represented by RefSeq NM_172084, and a nucleic acid containing a
CDS represented by RefSeq XM_005249861.
[0112] A typical example of human-derived "CaMKIV" includes a
protein specified under RefSeq NP_001735. Moreover, a typical
example of a human-derived nucleic acid encoding CaMKIV includes a
nucleic acid containing a CDS represented by RefSeq NM_001744.
[0113] Typical examples of human-derived "CaMKII.sigma." include a
protein specified under RefSeq NP_001212, a protein specified under
RefSeqNP_742112, a protein specified under RefSeq NP_742113, a
protein specified under RefSeq NP_742125, a protein specified under
RefSeq NP_742126, a protein specified under RefSeq NP_742127, and a
protein specified under RefSeq XP_005263312. Moreover, typical
examples of a human-derived nucleic acid encoding CaMKII.sigma.
include a nucleic acid containing a CDS represented by RefSeq
NM_001221, a nucleic acid containing a CDS represented by RefSeq
NM_172114, a nucleic acid containing a CDS represented by RefSeq
NM_172115, a nucleic acid containing a CDS represented by
RefSeqNM_172127, a nucleic acid containing a CDS represented by
RefSeq NM_172128, a nucleic acid containing a CDS represented by
RefSeq NM_172129, and a nucleic acid containing a CDS represented
by RefSeq XM_005263255.
[0114] A typical example of human-derived "CaMKII.alpha." includes
a protein specified under RefSeq NP_741960. Moreover, a typical
example of a human-derived nucleic acid encoding CaMKII.alpha.
includes a nucleic acid containing a CDS represented by RefSeq
NM_171825. "CSK" is a protein also referred to as casein kinase.
Examples thereof include CSKII.alpha. and CSKII subunit
.alpha..
[0115] Typical examples of human-derived "CSKII.alpha." include a
protein specified under RefSeq NP_001886, a protein specified under
RefSeq NP_808227, and a protein specified under RefSeq NP_808228.
Moreover, typical examples of a human-derived nucleic acid encoding
CSKII include a nucleic acid containing a CDS represented by RefSeq
NM_001895, a nucleic acid containing a CDS represented by RefSeq
NM_177559, and a nucleic acid containing a CDS represented by
RefSeq NM_177560.
[0116] A typical example of human-derived "CSKII subunit .alpha."
includes a protein specified under RefSeq NP_001887. Moreover, a
typical example of a human-derived nucleic acid encoding CSKII
subunit .alpha. includes a nucleic acid containing a CDS
represented by RefSeq NM_001896.
[0117] "Lyn" is a protein also referred to as Lyn tyrosine kinase.
Typical human-derived examples thereof include a protein specified
under RefSeq NP_001104567, a protein specified under
RefSeqNP_002341, a protein specified under RefSeq XP_005251289, and
a protein specified under RefSeq XP_005251290. Moreover, typical
examples of a human-derived nucleic acid encoding Lyn include a
nucleic acid containing a CDS represented by RefSeq NM_001111097, a
nucleic acid containing a CDS represented by RefSeq NM_002350, a
nucleic acid containing a CDS represented by RefSeq XM_005251232,
and a nucleic acid containing a CDS represented by RefSeq
XM_005251233.
[0118] "b-RAF" is a protein also referred to as b-RAF
serine/threonine kinase. A typical human-derived example thereof
includes a protein specified under RefSeqNP_004324. Moreover, a
typical example of a human-derived nucleic acid encoding b-RAF
includes a nucleic acid containing a CDS represented by RefSeq
NM_004333.4.
Advantageous Effects of Invention
[0119] The present invention makes it possible to diagnose before
the onset of Alzheimer's disease, and further to provide agents and
methods effective for treating the disease. Moreover, the present
invention makes it possible to diagnose frontotemporal lobar
degeneration before the onset, and further to provide agents and
methods effective for treating the disease.
BRIEF DESCRIPTION OF DRAWINGS
[0120] FIG. 1 is a diagram showing phosphoproteins which were
identified by two different approaches (hypothesis free approach
and A.beta. aggregation-linked approach) and whose expression
amounts were enhanced commonly in multiple Alzheimer's disease (AD)
model mice. In the figure, "MARCS" and "MARCKSL1" respectively
represent MARCKS and Marcksl1.
[0121] FIG. 2 is a schematic diagram showing a network constructed
of the 17 proteins shown in FIG. 1. Note that the 17 proteins are
proteins (AD core proteins) revealed to have phosphorylation levels
enhanced in the brains affected with Alzheimer's disease identified
in the present invention and to play central roles in a pathology
of the disease. Moreover, in the figure, lines (edges) connecting
the proteins (nodes) represent interactions therebetween.
[0122] FIG. 3 is a schematic diagram showing chronological changes
in phosphorylations of kinases and substrate proteins thereof in a
pre-onset stage of Alzheimer's disease. To be more specific, it is
shown that: at the ages of 1 to 3 months, the phosphorylations of
MARCKS, MARCKSL1, and SRRM2 in AD model mice (5.times.FAD mice) are
remarkably high in comparison with those of the wild type; at the
ages of 3 to 6 months, the phosphorylations of G3P, SYT1, SPTA2,
ADDB, NEUM, BASP1, and HSP90A in the AD model mice are remarkably
high in comparison with those of the wild type; and at the age of 6
months or later, the phosphorylations of CLH, NFH, NFL, and GPRIN1
in the AD model mice are remarkably high in comparison with those
of the wild type. Note that, in the 12-month-old AD model mice, no
onset (such as abnormal behavior) is observed. Moreover, in the
figure, lines (edges) connecting the proteins (nodes) represent
interactions therebetween. In the figure, regarding "PKC", see J
Biol Chem., 1994, Vol. 269, No. 30, pp. 19462 to 19465 and J
Neurochem., 1999, Vol. 73, Iss. 3, pp. 921 to 932. In addition,
regarding "CSKII", see J Biol Chem., 1993, Vol. 268, No. 9, pp.
6816 to 6822.
[0123] FIG. 4 shows photograph for illustrating the result of
administering a kinase inhibitor (Go697 or KN-93) or a Lyn kinase
activator (MLR1023) into 5.times.FAD mice (12 weeks old), and
observing dendritic spines/dendrites in layer 1 of the
retrosplenial cortex 36 hours and 60 hours thereafter. Note that
the observation results of administering DMSO (solvent alone) into
5.times.FAD mice and background mice thereof (B6/SJL (WT)) as
controls are also shown together.
[0124] FIG. 5 shows graphs for illustrating the result of
quantitatively analyzing the dendritic spine densities in the
5.times.FAD mice 36 hours and 60 hours after Go697, KN-93 or
MLR1023 was administered. To be more specific, the graphs show that
clearly the number of protrusions was descreased in the 5.times.FAD
mice, while the treatment with Go697, KN-93, or MLR1023 recovered
the decrease of spines in the 5.times.FAD mice. In the figure, A
shows the result of administering DMSO (solvent alone) into the
background (WT) mice, B shows the result of administering DMSO into
the 5.times.FAD mice, C shows the result of administering Go697
into the 5.times.FAD mice, D shows the result of administering
MLR1023 into the 5.times.FAD mice, and E shows the result of
administering KN-93 into the 5.times.FAD mice. Moreover, each bar
graph shows the average value+/-the standard error, and is provided
with numerical values indicating the number of samples in each
administration group. One and two asterisks respectively indicate
p<0.05 and p<0.01 Student's independent t-test (regarding the
representations in the figure, the same shall apply to FIGS. 6, 8,
9, and 11).
[0125] FIG. 6 shows graphs for illustrating the result of
quantitatively analyzing the spine type 36 hours and 60 hours after
Go697, KN-93, or MLR1023 was administered. To be more specific, the
graphs show that, regardless of the morphological type, the spine
densities were decreased in the 5.times.FAD mice, and that the
decreases were recovered by the treatment with Go697, KN-93, or
MLR1023.
[0126] FIG. 7 shows photographs illustrating the spine formation
and elimination in the 5.times.FAD mice 36 hours and 60 hours after
Go697, KN-93, or MLR1023 was administered. The arrow in the figure
after 36 hours (36 h) indicates the spine to be eliminated. The
arrows in the figure after 60 hours (60 h) indicate formed spines.
Note that the observation results of administering DMSO (solvent
alone) into the 5.times.FAD mice and the background mice (B6/SJL
(WT) as the controls are also shown together.
[0127] FIG. 8 shows graphs for illustrating the result of
quantitatively analyzing the dendritic spine dynamics in the
5.times.FAD mice 36 hours and 60 hours after Go697, KN-93, or
MLR1023 was administered. In the figure, the vertical axis
represents the number of formed spines, eliminated spines, or
stably remaining spines per 100 .mu.m of the dendritic shaft.
[0128] FIG. 9 shows graphs for illustrating the result of
quantitatively analyzing the dendritic spine dynamics in the
5.times.FAD mice36 hours and 60 hours after Go697, KN-93, or
MLR1023 was administered. In the figure, the vertical axis
represents the relative percentage of formed spines, eliminated
spines, or stably remaining spines.
[0129] FIG. 10 shows micrographs for illustrating the result of
analyzing the cerebral cortexes of WT/DMSO (the background mice
treated with DMSO alone), 5.times.FAD/DMSO (the 5.times.FAD mice
treated with DMSO alone), 5.times.FAD/Go (the 5.times.FAD mice
treated with Go697), 5.times.FAD/MLR (the 5.times.FAD mice treated
with MLR1023), and 5.times.FAD/KN-93 (the 5.times.FAD mice treated
with KN-93) by immunohistological staining using antibodies against
activated PKC.beta. (PKC.beta. pT642), activated PKC.delta.
(PKC.delta. pS643/676), and activated CamKII (CamKII pT286).
[0130] FIG. 11 shows graphs for illustrating the result of
quantitatively analyzing signals from each activated kinase in
neuronal somas or neuropils of the five types of mice shown in FIG.
10.
[0131] FIG. 12 shows photographs for illustrating the result of
injecting a lentiviral vector encoding shRNA against MARCKS into
layer 1 of the retrosplenial cortex, and observing dendritic
spines/dendrites at the site 4 days and 5 days later. Note that the
observation results of injecting scrambled shRNA into 5.times.FAD
mice and background mice thereof (B6/SJL (WT)) as controls are also
shown together.
[0132] FIG. 13 shows graphs for illustrating the result of
quantitatively analyzing the dendritic spine densities in the
5.times.FAD mice 4 days and 5 days after the shRNA against MARCKS
was injected. In the figure, A shows the result of injecting the
scrambled shRNA into the background (WT) mice, B shows the result
of injecting the scrambled shRNA into the 5.times.FAD mice, and C
shows the result of injecting the shRNA against MARCKS into the
5.times.FAD mice. Moreover, each bar graph shows the average
value+/-the standard error, and is provided with numerical values
(n=4) indicating the number of samples in each injection group. One
and two asterisks respectively indicate p<0.05 and p<0.01 in
Student's independent t-test (regarding the representations in the
figure, the same shall apply to FIGS. 14, 15, 17, and 18).
[0133] FIG. 14 shows graphs for illustrating the result of
quantitatively analyzing the spine type 4 days after the shRNA
against MARCKS was injected.
[0134] FIG. 15 shows graphs for illustrating the result of
quantitatively analyzing the spine type 5 days after the shRNA
against MARCKS was injected.
[0135] FIG. 16 shows photographs illustrating the spine formation
and elimination in the 5.times.FAD mice 4 days and 5 days after the
shRNA against MARCKS was injected. In the figure, the arrow
provided to the observation result after 4 days (4d) indicates the
spine to be eliminated, while the other arrows indicate formed
spines. Note that the observation result of injecting the scrambled
shRNA into the 5.times.FAD mice and the background mice (B6/SJL
(WT)) as the controls are also shown together.
[0136] FIG. 17 shows graphs for illustrating the result of
quantitatively analyzing the dendritic spine dynamics in the
5.times.FAD mice in which the shRNA against MARCKS was injected. In
the figure, the vertical axis represents the number of formed
spines, eliminated spines, or stably remaining spines per 100 .mu.m
of the dendritic shaft.
[0137] FIG. 18 shows graphs for illustrating the result of
quantitatively analyzing the dendritic spine dynamics in the
5.times.FAD mice in which the shRNA against MARCKS was injected. In
the figure, the vertical axis represents the relative percentage of
formed spines, eliminated spines, or stably remaining spines.
[0138] FIG. 19 shows photographs for illustrating the result of
treating mouse primary cortical nerve cells (E18) with a 10-.mu.M
amyloid .beta. protein (A.beta.1-42) in a medium for 6 hours in the
presence or absence of a b-raf kinase inhibitor, followed by
analysis by western blotting using an anti-phosphorylated tau
antibody. Note that, as the b-raf kinase inhibitor, PLX-4720 (PLX),
sorafenib (Sor), and GDC-0879 (GDC) were used, each of which was
added to the medium at the concentration of 1 .mu.M or 10
.mu.M.
[0139] FIG. 20 is a graph for illustrating the result of
quantitatively analyzing bands of the western blot shown in FIG.
19. In Student's independent t-test, one asterisk indicates
p<0.05 (n=3).
[0140] FIG. 21 is a schematic diagram showing a protocol for
analyzing the therapeutic effect of a b-raf inhibitor on the
behavioral phenotype of FTLD model mice.
[0141] FIG. 22 shows graphs for illustrating the result of
providing a b-raf inhibitor (vemurafenib) to FTLD model mice
(PGRN-KI mice), and evaluating the behavior of these mice by a
Morris water maze test. In the figure, the numbers shown in bars of
the graphs indicate the numbers of mice analyzed in each test.
Moreover, "-" shows the result (negative control) of mice to which
PBS was administered in place of vemurafenib.
[0142] FIG. 23 is a graph for illustrating the result of providing
vemurafenib to the PGRN-KI mice, and evaluating the behavior of
these mice by a fear-conditioning test (the representations in the
figure are the same as FIG. 22).
[0143] FIG. 24 is a schematic diagram showing a protocol analyzing
the therapeutic effect of a TNF signal transduction inhibitor on
the behavioral phenotype of the FTLD model mice.
[0144] FIG. 25 shows graphs for illustrating the result of
providing a TNF signal transduction inhibitor (thalidomide) to the
FTLD model mice (PGRN-KI mice), and evaluating the behavior of
these mice by the Morris water maze test. In the figure, the
numbers shown in bars of the graphs indicate the numbers of mice
analyzed in each test. Moreover, "-" shows the result (negative
control) of mice to which PBS was administered in place of
thalidomide.
[0145] FIG. 26 is a graph for illustrating the result of providing
thalidomide to the PGRN-KI mice, and evaluating the behavior of
these mice by the fear-conditioning test (the representations in
the figure are the same as FIG. 25).
[0146] FIG. 27 is a figure for illustrating the result of western
blot analysis on the cerebral cortexes of wild-type mice (WT) and
the PGRN-KI mice to which vemurafenib was administered. In the
figure, the upper panel shows photographs for illustrating the
western blot analysis result. "Anti-p-Braf" and "Anti-p-PKC"
respectively show the analysis result of phosphorylated b-RAF
protein and the analysis result of phosphorylated PKC protein.
"Anti-GAPDH" shows the result of detecting a GAPDH protein as an
internal standard. Moreover, in the figure, the lower two panels
are graphs for illustrating the western blot analysis result: the
left side shows the relative value of the phosphorylated b-RAF
protein amount based on the GAPDH amount, and the right side shows
the relative value of the phosphorylated PKC protein amount based
on the GAPDH amount. The significant differences were evaluated
based on the P-value calculated by Student's independent
t-test.
[0147] FIG. 28 is a figure for illustrating the result of western
blot analysis on the cerebral cortexes of the PGRN-KI mice to which
thalidomide was administered. In the figure, the left panel shows
photographs for illustrating the western blot analysis result.
"Anti-p-Braf", "Anti-Braf", and "Anti-p-PKC" respectively show the
analysis result of phosphorylated b-raf protein, the analysis
result of b-raf protein, and the analysis result of phosphorylated
PKC protein. "Anti-GAPDH" shows the result of detecting a GAPDH
protein as an internal standard. Moreover, in the figure, the right
panel shows graphs for illustrating the western blot analysis
result. "p-B-raf/Braf" shows the relative value of the
phosphorylated b-raf protein amount based on a total b-raf protein
amount, "p-B-raf/GAPDH" shows the relative value of the
phosphorylated b-raf protein amount based on the GAPDH amount, and
"p-PKC/GAPDH" shows the relative value of the phosphorylated PKC
protein amount based on the GAPDH amount. Further, in the figure,
one asterisk indicates p<0.05 in Student's independent t-test,
and two asterisks indicate p<0.01 in Student's independent
t-test.
[0148] FIG. 29 is a figure for illustrating the result of spine
static analysis on the retrosplenial cortexes (RSD) of the FTLD
model mice (PGRN-KI mice) and wild-type mice (WT). In the figure,
the left panel shows photographs of spines observed with a
two-photon microscope. In the figure, graphs in the right panel
show the number of spines (the number of spines per 1 .mu.m of the
dendrite), spine length, spine head diameter, and spine volume
measured by the two-photon microscope observation. Moreover, in
each graph, the left bar shows the observation result of the
wild-type mice, and the right bar shows the observation result of
the PGRN-KI mice. In the figure, two asterisks indicate p<0.01
in Student's independent t-test.
[0149] FIG. 30 is a figure for illustrating the result of spine
dynamic analysis on the retrosplenial cortexes (RSD) of the FTLD
model mice (PGRN-KI mice) and wild-type mice (WT). In the figure,
the left panel shows photographs of spines observed with a
two-photon microscope. In the photographs, the upward arrows
indicate spines to be eliminated, and the downward arrows indicate
produced spines. The right panel shows the number of spines
produced, the number of spines eliminated, and the number of spines
stably remaining detected by the two-photon microscope observation.
Moreover, in each graph, the left bar shows the observation result
of the wild-type mice, and the right bar shows the observation
result of the PGRN-KI mice.
[0150] FIG. 31 is a figure for illustrating the result of spine
static analysis on the retrosplenial cortexes (RSD) of the PGRN-KI
mice (KI) to which the b-raf inhibitor (vemurafenib) or PBS was
administered. In the figure, the left panel shows photographs of
spines observed with a two-photon microscope. In the figure, graphs
in the right panel show the number of spines (the number of spines
per 1 .mu.m of the dendrite), spine length, spine head diameter,
and spine volume measured by the two-photon microscope observation.
Moreover, in each graph, the left bar shows the observation result
of the PBS-administered PGRN-KI mice (KI+PBS), and the right bar
shows the observation result of the vemurafenib-administered
PGRN-KI mice (KI+vemurafenib). In the figure, one asterisk
indicates p<0.05 in Student's independent t-test.
[0151] FIG. 32 is a figure for illustrating the result of spine
static analysis on the retrosplenial cortexes (RSD) of the PGRN-KI
mice (KI) in which shRNA against tau (Sh-Tau) or scrambled shRNA
(Sh-scrambled) was injected. In the figure, the left panel shows
photographs of spines observed with a two-photon microscope. In the
figure, graphs in the right panel show the number of spines (the
number of spines per 1 .mu.m of the dendrite), spine length, spine
head diameter, and spine volume measured by the two-photon
microscope observation. Moreover, in each graph, the left bar shows
the observation result of the scrambled shRNA-injected PGRN-KI mice
(KI+Sh-scrambled), and the right bar shows the observation result
of the Sh-Tau-injected PGRN-KI mice (KI+Sh-Tau). In the figure, one
asterisk indicates p<0.05 in Student's independent t-test.
[0152] FIG. 33 shows graphs for illustrating the result of spine
dynamics analysis on the retrosplenial cortexes of the FTLD model
mice (PGRN-KI mice). In the figure, the left graph shows the result
of administering vemurafenib to the PGRN-KI mice (the number of
analyses: three mice, the left bars in the graph) or the result of
administering PBS to the PGRN-KI mice (the number of analyses: four
mice, the right bars in the graph). In the figure, the right graph
shows the result of injecting scrambled shRNA into the PGRN-KI mice
(the number of analyses: four mice, the left bars in the graph) or
the result of injecting shRNA against tau into the PGRN-KI mice
(the number of analyses: four mice, the right bars in the
graph).
DESCRIPTION OF EMBODIMENTS
[0153] <Method 1 for Diagnosing Alzheimer's Disease>
[0154] As will be described later in Examples, it has been revealed
that phosphorylations of MARCKS, Marcksl1, SRRM2, SPTA2, ADDB,
NEUM, BASP1, SYT1, G3P, HS90A, CLH, NFH, NFL, GPRIN1, ACON, ATPA,
and ATPB are commonly enhanced in multiple Alzheimer's disease
model mice before the onset of the disease. Thus, the present
invention provides a method for diagnosing Alzheimer's disease
based on the phosphorylation of these proteins, the method
comprising the following the steps (i) to (iii): [0155] (i) a step
of detecting, in a test subject, a phosphorylation of at least one
substrate protein selected from the group consisting of MARCKS,
Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A, CLH,
NFH, NFL, GPRIN1, ACON, ATPA, and ATPB; [0156] (ii) a step of
comparing the phosphorylation with a phosphorylation of a substrate
protein in a normal subject; and [0157] (iii) a step of determining
that the test subject is affected with Alzheimer's disease or has a
risk of developing Alzheimer's disease if the phosphorylation of
the substrate protein in the test subject is higher than the
phosphorylation of the substrate protein in the normal subject as a
result of the comparison.
[0158] In this diagnosis method, the substrate proteins such as
MARCKS are not limited respectively to the proteins having the
amino acid sequences listed as the typical examples described
above, and naturally-occurring mutants thereof can also be
targeted. Moreover, in a case where multiple sites (amino acid
residues) are phosphorylated in one substrate protein, the
phosphorylation of at least one site of the protein should be
detected. Nevertheless, from the viewpoint of further increasing
the diagnosis precision, it is preferable to detect all the
phosphorylation sites of the protein.
[0159] In the case of human, examples of the phosphorylation site
in MARCKS to be detected by the present invention include serine at
position 26, serine at position 27, serine at position 29, serine
at position 118, serine at position 128, serine at position 131,
serine at position 132, serine at position 134, serine at position
135, serine at position 145, serine at position 147, threonine at
position 150, serine at position 170, and serine at position 322.
Examples thereof in Marcksl1 include serine at position 22,
threonine at position 85, serine at position 104, threonine at
position 148, serine at position 151, serine at position 180, and
serine at position 184. Examples thereof in SRRM2 include serine at
position 1102, serine at position 1320, serine at position 1348,
serine at position 1383, serine at position 1403, serine at
position 1404, serine at position 2398, serine at position 2132,
serine at position 2449, serine at position 2581, threonine at
position 1492, and threonine at position 2397. Examples thereof in
SPTA2 include serine at position 1031 and serine at position 1217.
Examples thereof in ADDB include serine at position 60, serine at
position 62, serine at position 532, serine at position 592, serine
at position 600, serine at position 617, serine at position 693,
and serine at position 701. Examples thereof in NEUM include serine
at position 151, threonine at position 181, and serine at position
203. Examples thereof in BASP1 include threonine at position 31,
threonine at position 36, serine at position 132, serine at
position 195, and serine at position 219. Examples thereof in SYT1
include threonine at position 126 and threonine at position 129.
Examples thereof in G3P include threonine at position 184 and
threonine at position 211. Examples thereof in HS90A include serine
at position 231 and serine at position 263. Examples thereof in NFH
include serine at position 503, serine at position 540, serine at
position 660, serine at position 730, serine at position 769,
serine at position 801, and serine at position 828. An example
thereof in NFL includes serine at position 472. Examples thereof in
GPRIN1 include serine at position 776, serine at position 799,
serine at position 850, serine at position 853, and threonine at
position 877.
[0160] Note that these phosphorylation sites are sites in
Alzheimer's disease model mice where phosphorylation levels were
changed and identified in Example 5 to be described later, and
converted to corresponding human sites (regarding the
correspondence between human and mouse at each phosphorylation
site, see PhosphoSite Plus
(http://www.phosphosite.org/homeAction.do)). Additionally, in the
present invention, the term "phosphorylation site" means a site
having at least 3 amino acids including one amino acid before and
one amino acid after a phosphorylated amino acid in a
phosphorylated protein such as the substrate protein.
[0161] In the diagnosis method, in a case where a test subject
whose substrate protein phosphorylation is to be detected is a
specimen (such as body fluid, tissue, cell) isolated from a body of
an animal including human, that is, where the diagnosis method is
an in vitro method, an example of the detection method in the step
(i) includes a mass spectrometry method as will be described later
in Examples, that is, a method in which phosphopolypeptides are
extracted from the specimen, labeled, and analyzed by 2D LC MS/MS.
Such a detection by a mass spectrometry method is preferable from
the viewpoint that multiple phosphorylations of multiple substrate
proteins can be comprehensively detected, consequently further
increasing the diagnosis precision.
[0162] Moreover, the in vitro method includes a detection method
using an antibody capable of specifically binding to a
phosphorylation site of a substrate protein, for example,
immunohistochemical staining, immunoelectron microscopy, and
immunoassays (such as enzyme immunoassay (ELISA, EIA), fluorescent
immunoassay, radioimmunoassay (RIA), immunochromatography, and
western blot method). Further, the example also includes a method
utilizing a detector (for example, BIAcore (manufactured by GE
Healthcare)) based on the surface plasmon resonance phenomenon
using a thin metal film on which a compound capable of specifically
binding to a phosphorylation site of a substrate protein is
immobilized. Regarding the antibody and the compound, see the
description of <Diagnostic Agent Against Alzheimer's Disease>
to be Described Later.
[0163] Meanwhile, in the diagnosis method, in a case where a test
subject whose substrate protein phosphorylation is to be detected
is a body of an animal including human, that is, where the
diagnosis method is an in vivo method, examples of the detection
method in the step (i) include bioimaging techniques (computerized
axial tomographies (CAT, CT), magnetic resonance imaging (MRI),
positron emission tomography (PET), single-photon emission computed
tomography (SPECT)). More concretely, the detection can be
performed with reference to the techniques described in
International Application Japanese-Phase Publication Nos.
2004-513123, 2004-530408, and 2002-514610, Japanese Unexamined
Patent Application Publication No. 2011-95273, International
Application Japanese-Phase Publication Nos. 2001-527509, Hei
9-501419, Hei 9-505799, and Hei 8-509226. Nevertheless, the
embodiment of the diagnosis method of the present invention is not
limited thereto.
[0164] In the bioimaging techniques, a compound capable of
specifically binding to a phosphorylation site of a substrate
protein is introduced into the body of a test subject. The
introduction method is not particularly limited, and examples
thereof include intravenous administration, intraarterial
administration, intraperitoneal administration, subcutaneous
administration, intradermal administration, tracheobronchial
administration, rectal administration and intramuscular
administration, administration by transfusion, and direct
administration into a target site (such as brain). The direct
administration into a target site can be achieved by employing, for
example, cannula (catheter), surgical incision, or the like.
Regarding the compound, see the description of <Diagnostic Agent
against Alzheimer's Disease> to be described later.
[0165] In addition, as will be described later in Examples, the
substrate proteins to be detected in the method for diagnosing
Alzheimer's disease are classified into three according to the
pattern of the chronological change in phosphorylation. To be more
specific, examples of the substrate protein whose phosphorylation
is enhanced the most at an initial phase of a pre-onset stage of
Alzheimer's disease include MARCKS, Marcksl1, and SRRM2; examples
of the substrate protein whose phosphorylation is enhanced the most
at a mid phase of the pre-onset stage of Alzheimer's disease
include SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, and HS90A; and
examples of the substrate protein whose phosphorylation is enhanced
the most at a late phase of the pre-onset stage of Alzheimer's
disease include CLH, NFH, NFL, and GPRIN1. Thus, in the step (iii),
if the phosphorylations of at least two substrate proteins among
MARCKS, Marcksl1, and SRRM2 (more preferably the phosphorylations
of all the three substrate proteins) are higher than those in a
normal subject, the test subject can be determined to be at the
initial phase before the onset of Alzheimer's disease. Moreover, if
the phosphorylations of at least two substrate proteins among
SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, and HS90A (more preferably the
phosphorylations of three substrate proteins, the phosphorylations
of four substrate proteins, the phosphorylations of five substrate
proteins, furthermore preferably the phosphorylations of six
substrate proteins, and particularly preferably the
phosphorylations of all the seven substrate proteins) are higher
than those in a normal subject, the test subject can be determined
to be at the mid phase before the onset of Alzheimer's disease.
Further, if the phosphorylations of at least two substrate proteins
among CLH, NFH, NFL, and GPRIN1 (more preferably the
phosphorylations of three substrate proteins, furthermore
preferably the phosphorylations of all the four substrate proteins)
are higher than those in a normal subject, the test subject can be
determined to be at the late phase before the onset of Alzheimer's
disease.
[0166] <Method 2 for Diagnosing Alzheimer's Disease>
[0167] In addition, as will be described later in Examples, it has
also been revealed that kinase proteins which phosphorylate the
substrate proteins such as MARCKS are activated in the Alzheimer's
disease model mice before the onset of the disease. Thus, the
present invention also provides, as a second embodiment of the
method for diagnosing Alzheimer's disease, a method comprising the
following the steps (i) to (iii): [0168] (i) a step of detecting an
activity or expression of a kinase protein in a test subject;
[0169] (ii) a step of comparing the activity or expression with an
activity or expression of a kinase protein in a normal subject; and
[0170] (iii) a step of determining that the test subject is
affected with Alzheimer's disease or has a risk of developing
Alzheimer's disease if the activity or expression of the kinase
protein in the test subject is higher than the activity or
expression of the kinase protein in the normal subject as a result
of the comparison, wherein [0171] the kinase protein is at least
one kinase protein selected from the group consisting of PKC, CaMK,
CSK, Lyn, and b-RAF.
[0172] In this diagnosis method, the kinase proteins such as PKC
are not limited respectively to the proteins having the amino acid
sequences listed as the typical examples described above, and
naturally-occurring mutants thereof can also be targeted. Moreover,
the "activity" of the kinase proteins to be detected means an
activity (kinase activity) of directly or indirectly
phosphorylating the substrate protein. Further, since a kinase
activity correlates with an amount of a kinase protein expressed,
particularly an amount of an activated kinase protein expressed,
the amount of a kinase protein expressed, preferably the amount of
an activated kinase protein expressed, can also be the target of
the detection by the diagnosis method in place of the kinase
activity.
[0173] In the present invention, the "activated kinase protein"
means a kinase protein in a state where the kinase protein is
capable of phosphorylating the substrate protein. An example
thereof includes a phosphorylated kinase protein. More concretely,
the examples of the activated kinase protein include PKC.beta.
having threonine at position 642 phosphorylated, PKC.alpha. having
threonine at position 638 phosphorylated, PKC.lamda./.left
brkt-bot. having threonine at position 403 phosphorylated,
PKC.delta. having serine at position 643 phosphorylated, PKC.xi.
having threonine at position 410 phosphorylated, PKC.xi. having
tyrosine at position 417 phosphorylated, CaMKI having serine at
position 177 phosphorylated, CaMKII.beta. having threonine at
position 287 phosphorylated, CaMKIV having threonine at position
200 phosphorylated, CaMKII.sigma. having threonine at position 287
phosphorylated, CaMKII.alpha. having threonine at position 286
phosphorylated, CSKII.alpha. having threonine at position 360
phosphorylated, CSKII.alpha. having serine at position 362
phosphorylated, Lyn having tyrosine at position 397 phosphorylated,
b-RAF having serine at position 365 phosphorylated, b-RAF having
serine at position 446 phosphorylated, b-RAF having serine at
position 579 phosphorylated, b-RAF having threonine at position 599
phosphorylated, b-RAF having serine at position 602 phosphorylated,
b-RAF having serine at position 729 phosphorylated, and b-RAF
having serine at position 732 phosphorylated.
[0174] In the case where the diagnosis method is an in vitro
method, the detection of the activity can be performed, for
example, by adding a substrate and a radiolabeled phosphate to the
specimen or a protein liquid extract thereof, and detecting the
incorporation of the phosphate into the substrate. The
incorporation of the phosphate into the substrate can be detected
with a scintillation counter, by autoradiography, or other means.
Alternatively, without using a radioactive label, the activity can
also be detected by treating the substrate with the specimen or a
protein liquid extract thereof, and detecting an increase in the
molecular weight of the substrate after the treatment. The
detection of an increase in the molecular weight can be performed,
for example, by detecting a change in mobility of the substrate in
polyacrylamide gel electrophoresis. Further, the polyacrylamide gel
after the electrophoresis may be transferred to a membrane such as
PVDF for the detection by a western blot method using an antibody
capable of specifically binding to a phosphorylation site of the
substrate. Note that examples of the substrate include known
substrate proteins of the targeted kinase proteins, and partial
peptides thereof containing a site to be phosphorylated
(phosphorylated site).
[0175] Examples of the known substrate proteins of PKC include
MARCKS, Marcksl1, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, and HSP90A.
More concretely, examples of the substrate proteins of PKC.beta.
include SPTA2, MARCKS, and NEUM; examples of the substrate protein
of PKC.alpha. include MARCKS and HSP90A; an example of the
substrate protein of PKC.xi. includes Marcksl1; examples of the
substrate protein of PKC.lamda./.left brkt-bot. include G3P and
HSP90A; and examples of the substrate protein of PKC.delta. include
ADDB, NEUM, and HSP90A.
[0176] Examples of the known substrate proteins of CaMK include
SPTA2 and G3P. More concretely, an example of the substrate protein
of CaMKI includes G3P; an example of the substrate protein of
CaMKII.beta. includes G3P; an example of the substrate protein of
CaMKIV includes G3P; and an example of the substrate protein of
CaMKII.delta. includes SPTA2.
[0177] Examples of the known substrate proteins of CSK include
NEUM, SYT1, and HSP90A. More concretely, an example of the
substrate protein of CSKII subunit .alpha. includes HSP90A; and
examples of the substrate protein of CSKII include NEUM and
HSP90A.
[0178] An example of the known substrate protein of Lyn includes
G3P. Moreover, examples of the substrate protein of b-RAF include
MEK1, ERK1, and tau.
[0179] Moreover, in the in vitro method, examples of the method for
detecting the expression amount include, as in the case of the
detection of substrate protein phosphorylation described above, a
mass spectrometry method, a detection method using an antibody
capable of specifically binding to a kinase protein (preferably,
activated kinase protein), and a method utilizing a detector based
on the surface plasmon resonance phenomenon using a thin metal film
on which a compound capable of specifically binding to a kinase
protein (preferably, activated kinase protein) is immobilized.
Regarding the antibody and the compound, see the description of
<Diagnostic Agent against Alzheimer's Disease> to be
described later.
[0180] In the case where the diagnosis method is an in vivo method,
the detection of the activity can be performed by detecting the
substrate protein phosphorylation attributable to the activity. To
be more specific, bioimaging techniques aiming at the detection of
substrate protein phosphorylation described above can be suitably
used.
[0181] Moreover, in the case where the diagnosis method is an in
vivo method, the detection of the expression amount can be
performed as in the case of the detection of substrate protein
phosphorylation described above, for example, by utilizing
bioimaging techniques using a compound capable of specifically
binding to a kinase protein (preferably, activated kinase protein).
Regarding the compound, see the description of <Diagnostic Agent
against Alzheimer's Disease> to be described later.
[0182] Hereinabove, preferred embodiments of the diagnosis method
of the present invention have been described. In addition, such a
diagnosis is normally conducted by a doctor (including one
instructed by a doctor). The data on the phosphorylation of the
substrate protein or the activity or expression of the kinase
protein in the test subject obtained by the diagnosis method of the
present invention is useful in the diagnosis by a doctor. Thus, the
method of the present invention can also be described as a method
for collecting and presenting such data useful in a diagnosis by a
doctor.
[0183] Moreover, the present invention makes it possible to
determine that one is affected with Alzheimer's disease or has a
risk of developing Alzheimer's disease. In this manner, enabling
judgment of Alzheimer's disease affection or the like at an early
stage leads to an expectation that treatment methods for
suppressing a pathology of Alzheimer's disease (immunotherapy, a
method for administering an agent for suppressing a pathology of
Alzheimer's disease) will be effective.
[0184] Thus, the present invention also makes it possible to
provide a method for treating Alzheimer's disease, the method
comprising: a step of administering an agent for suppressing a
pathology of Alzheimer's disease to a test subject determined to be
affected with Alzheimer's disease or have a risk of developing
Alzheimer's disease by the method for diagnosing Alzheimer's
disease of the present invention, and/or a step of conducting an
immunotherapy for the test subject.
[0185] Examples of the immunotherapy include active immunotherapy
(vaccine therapy) using a partial peptide of amyloid .beta. to
suppress amyloid .beta. aggregation, and passive immunotherapy in
which an antibody against amyloid .beta. is administered.
[0186] Additionally, examples of the agent administered to suppress
a pathology of Alzheimer's disease include agents for suppressing
amyloid (production (such as .gamma.-secretase modulators (GSM),
.gamma.-secretase modulator inhibitors (GSI), nonsteroidal
anti-inflammatory drugs), agents for suppressing amyloid .beta.
aggregation (such as curcumin, polysulfuric acid compound,
clioquinol), agents for suppressing tau aggregation (such as
aminothienopyridazine, cyanine dye, methylene blue),
neuroprotective drugs (such as dimebon), cholinesterase inhibitors
(such as donepezil), acetylcholinesterase inhibitors (such as
galantamine), and NMDA glutamate receptor inhibitors (such as
memantine).
[0187] Further, as will be described later, it is also possible to
suitably use, as the agent for suppressing a pathology of
Alzheimer's disease, an agent for treating Alzheimer's disease, the
agent comprising any one of: a compound capable of suppressing a
phosphorylation of at least one substrate protein selected from the
group consisting of MARCKS, Marcksl1, SRRM2, SPTA2, ADDB, NEUM,
BASP1, SYT1, G3P, HS90A, CLH, NFH, NFL, GPRIN1, ACON, ATPA, and
ATPB; a compound capable of suppressing an activity or expression
of at least one kinase protein selected from the group consisting
of PKC, CaMK, CSK, Lyn, and b-RAF; a compound capable of activating
Lyn; and a compound capable of suppressing a binding of at least
one substrate protein selected from the group consisting of MARCKS,
Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A, CLH,
NFH, NFL, GPRIN1, ACON, ATPA, and ATPB to at least one kinase
protein selected from the group consisting of PKC, CaMK, CSK, Lyn,
and b-RAF.
[0188] <Diagnostic Agent Against Alzheimer's Disease>
[0189] As described above, it has been revealed that the
phosphorylations of MARCKS and the like are commonly enhanced in
the multiple Alzheimer's disease model mice before the onset of the
disease. Thus, the present invention provides an agent for
diagnosing Alzheimer's disease, the agent comprising a compound
having an activity of binding to a phosphorylation site of at least
one substrate protein selected from the group consisting of MARCKS,
Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A, CLH,
NFH, NFL, GPRIN1, ACON, ATPA, and ATPB (hereinafter, the agent for
diagnosing Alzheimer's disease may also be referred to as
"Alzheimer's disease diagnostic agent").
[0190] As described above, it has been revealed that the kinase
proteins which phosphorylate the substrate proteins such as MARCKS
are also activated in the Alzheimer's disease model mice before the
onset of the disease. Thus, the present invention provides an agent
for diagnosing Alzheimer's disease, the agent comprising a compound
having an activity of binding to at least one kinase protein
selected from the group consisting of PKC, CaMK, CSK, Lyn, and
b-RAF. Further, an example of a preferred embodiment of the agent
includes an agent for diagnosing Alzheimer's disease, the agent
comprising a compound having an activity of binding to at least one
activated kinase protein selected from the group consisting of PKC,
CaMK, CSK, Lyn, and b-RAF.
[0191] As in the diagnosis method described above, the substrate
proteins and the kinase proteins targeted by the diagnostic agents
of the present invention are not limited respectively to the
proteins having the amino acid sequences listed as the typical
examples described above, and naturally-occurring mutants thereof
can also be targeted.
[0192] The "compound having an activity of binding to the
phosphorylation site of the substrate protein" and the "compound
having an activity of binding to the kinase protein" are not
particularly limited, and may be known compounds or may be ones
identified by screening to be described later. Examples of such
compounds include antibodies capable of binding to the
phosphorylation site of the substrate protein or the kinase
protein, and low-molecular-weight compounds capable of binding to
the phosphorylation site of the substrate protein or the kinase
protein.
[0193] An "antibody" in the present invention may be a polyclonal
antibody, a monoclonal antibody, or a functional fragment of an
antibody. The antibody includes all classes and subclasses of
immunoglobulins. The "functional fragment" of an antibody means a
part (partial fragment) of an antibody and capable of specifically
recognizing an antigen thereof. Concretely, examples thereof
include Fab, Fab', F(ab')2, a variable region fragment (Fv), a
disulfide bonded Fv, a single chain Fv (scFv), a sc(Fv)2, a
diabody, a polyspecific antibody, polymers thereof, and the like.
Moreover, the antibody includes a chimeric antibody, a humanized
antibody, a human antibody, and functional fragments of these
antibodies. Further, the amino acid sequences of these antibodies
may undergo alteration, modification, or the like as necessary.
Those skilled in the art can prepare such antibodies as appropriate
by known antibody preparation methods. Furthermore, in a case where
the agents for diagnosing Alzheimer's disease of the present
invention or agents for treating the disease to be described later
are to be introduced into human, preferable among these antibodies
are a humanized antibody, a human antibody, and functional
fragments of these antibodies, from the viewpoint that an
immunoreaction hardly occurs with the introduced antibody.
[0194] In addition, the "compound having an activity of binding to
the phosphorylation site of the substrate protein" and the
"compound having an activity of binding to the kinase protein"
preferably have a labeling substance bound thereto for the
detection by the above-described detection methods using an
antibody, bioimaging techniques, and the like. The labeling
substance is selected as appropriate in accordance with the type of
the detection method employed and the like. Examples thereof
include radioactive labeling substances, fluorescent labeling
substances, paramagnetic labeling substances, superparamagnetic
labeling substances, and enzyme labeling substances. Moreover, such
labeling substances may be bound to the molecules directly or
indirectly. Examples of the indirect binding include bindings
utilizing a secondary antibody to which a labeling substance is
bound, or a polymer (such as Protein A, Protein B) to which a
labeling substance is bound.
[0195] The agents of the present invention may comprise, in
addition to the compounds, other pharmacologically acceptable
ingredients. Examples of such other ingredients include a carrier,
an excipient, a disintegrator, a buffer, an emulsifier, a
suspension, a stabilizer, a preservative, an antiseptic, and a
physiological salt. As the excipient, lactose, starch, sorbitol,
D-mannitol, white sugar, or the like can be used. As the
disintegrator, starch, carboxymethyl cellulose, calcium carbonate,
or the like can be used. As the buffer, a phosphate, a citrate, an
acetate, or the like can be used. As the emulsifier, gum arabic,
sodium alginate, traganth, or the like can be used. As the
suspension, glyceryl monostearate, aluminium monostearate, methyl
cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium
lauryl sulfate, or the like can be used. As the stabilizer,
propylene glycol, diethylin sulfite, ascorbic acid, or the like can
be used. As the preservative, phenol, benzalkonium chloride, benzyl
alcohol, chlorobutanol, methylparaben, or the like can be used. As
the antiseptic, sodium azide, benzalkonium chloride,
para-oxybenzoic acid, chlorobutanol, or the like can be used.
[0196] When the diagnostic agents of the present invention are used
in vivo, the administration method into the body of a test subject
is as described above in the description of <Method for
diagnosing Alzheimer's disease>. Moreover, those skilled in the
art can adjust an amount of the diagnostic agents of the present
invention administered and the number of administrations as
appropriate depending on the type of the compounds, the body weight
of a test subject, and the like. The number of administrations can
be adjusted as appropriate depending on the administration amount,
the administration route, and the like.
[0197] A product of the diagnostic agents of the present invention
or a manual thereof may be provided with an indication stating that
the product is used for diagnosing the target disease. Herein, "a
product or a manual provided with an indication" means that the
indication is provided to a main body, a container, a package, or
the like of the product, or the indication is provided to a manual,
a package insert, an advertisement, other printed matters, or the
like in which information on the product is disclosed.
[0198] <Screening Method for Alzheimer's Disease Diagnostic
Agent Candidate Compound>
[0199] As described above, it has been revealed that the
phosphorylations of the substrate proteins such as MARCKS are
commonly enhanced in the multiple Alzheimer's disease model mice
before the onset of the disease. Further, it has also been revealed
that the kinase proteins which phosphorylate these substrate
proteins are activated in the Alzheimer's disease model mice before
the onset of the disease. Thus, based on such findings, the present
invention makes it possible to provide two embodiments of a
screening method for a candidate compound for diagnosing
Alzheimer's disease described below.
(1) A method comprising the steps of: [0200] bringing a test
compound into contact with a phosphorylation site of at least one
substrate protein selected from the group consisting of MARCKS,
Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A, CLH,
NFH, NFL, GPRIN1, ACON, ATPA, and ATPB; and [0201] selecting the
compound if the compound binds to the phosphorylation site. (2) A
method comprising the steps of: [0202] bringing a test compound
into contact with at least one kinase protein selected from the
group consisting of PKC, CaMK, CSK, Lyn, and b-RAF; and [0203]
selecting the compound if the compound binds to the kinase
protein.
[0204] The test compound used in the screening methods of the
present invention is not particularly limited. Examples thereof
include expression products of gene libraries, synthetic
low-molecular-weight compound libraries, peptide libraries,
antibodies, substances released from bacteria; liquid extracts and
culture supernants of cells (microorganisms, plant cells, animal
cells), purified or partially purified polypeptides, extracts
derived from marine organisms, plants, or animals, soils, and
random phage peptide display libraries.
[0205] The substrate proteins such as MARCKS and the kinase
proteins such as PKC used in these screening methods are as
described above in the description of <Diagnostic Agent against
Alzheimer's Disease>. Nevertheless, the kinase protein is
preferably an activated kinase protein.
[0206] Moreover, from the viewpoint of the easiness of the
detection of the binding, a reporter protein (for example, GFP,
luciferase), a tag protein for purification (for example, histidine
tag, FLAG tag, GST tag), or the like may be added to these
proteins. Further, these proteins may be partial peptides, but the
substrate proteins and the activated kinase protein need to contain
at least a phosphorylation site(s).
[0207] Moreover, the detection of the binding to these proteins is
not particularly limited, and can be performed by selecting a known
method as appropriate. Examples of the known method include a
co-immunoprecipitation method, an ELISA method, a method using a
detector based on the surface plasmon resonance phenomenon, and a
method based on FRET (fluorescence resonance energy transfer).
[0208] <Alzheimer's Disease Therapeutic Agent 1>
[0209] As will be described later in Examples, it has been revealed
that the phosphorylations of the substrate proteins such as MARCKS
are commonly enhanced in the multiple Alzheimer's disease model
mice before the onset of the disease. Further, it has also been
found that suppressing expressions of the substrate proteins using
shRNA successfully suppresses a pathology (abnormal spine
formation) in the Alzheimer's disease model mice.
[0210] Thus, the present invention provides an agent for treating
Alzheimer's disease, the agent comprising a compound capable of
suppressing a phosphorylation of at least one substrate protein
selected from the group consisting of MARCKS, Marcksl1, SRRM2,
SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A, CLH, NFH, NFL, GPRIN1,
ACON, ATPA, and ATPB (hereinafter, the agent for treating
Alzheimer's disease may also be referred to as simply "therapeutic
agent").
[0211] The substrate proteins such as MARCKS targeted by the
therapeutic agent of the present invention are as described above
in the description of <Diagnostic Agent against Alzheimer's
Disease>. In addition, "suppressing" of a phosphorylation of a
substrate protein and related terms mean to include not only
complete suppression (inhibition) but also partial suppression of
the phosphorylation.
[0212] Moreover, the suppression of the phosphorylation of the
substrate protein can also be achieved by suppressing the
expression of the substrate protein per se. Thus, the "compound
capable of suppressing the phosphorylation of the substrate
protein" also includes a "compound capable of suppressing the
expression of the substrate protein."
[0213] The "compound capable of suppressing the phosphorylation of
the substrate protein" is not particularly limited, and may be a
known compound or may be one identified by the screening to be
described later. Examples of such a compound include antibodies
capable of binding to a phosphorylated site of the substrate
protein, low-molecular-weight compounds capable of binding to a
phosphorylated site of the substrate protein, RNAs capable of
binding to a transcription product of a gene encoding the substrate
protein, and peptides having a dominant negative phenotype against
the substrate protein. Regarding the antibodies, see <Diagnostic
Agent against Alzheimer's Disease>. Regarding such "RNAs capable
of binding to a transcription product of a gene encoding a protein"
and "peptides having a dominant negative phenotype against a
protein," see the description to be described later.
[0214] Note that, in the present invention, the term
"phosphorylated site" means a site having at least 3 amino acids
including one amino acid before and one amino acid after a
phosphorylated amino acid in a protein obtained as a result of
phosphorylation such as the substrate protein.
[0215] <Alzheimer's Disease Therapeutic Agent 2>
[0216] As will be described later in Examples, it has also been
revealed that the kinase proteins which phosphorylate the substrate
proteins are activated in the Alzheimer's disease model mice before
the onset of the disease. Further, it has also been found that
suppressing the activations of the kinase proteins by using an
inhibitor against the proteins successfully suppresses the
pathology in the Alzheimer's disease model mice.
[0217] Thus, the present invention also provides, as a second
embodiment of the therapeutic agent against Alzheimer's disease, an
agent comprising a compound capable of suppressing an activity or
expression of at least one kinase protein selected from the group
consisting of PKC, CaMK, CSK, Lyn, and b-RAF.
[0218] The kinase proteins such as PKC targeted by the therapeutic
agent of the present invention are as described above in the
description of <Diagnostic Agent against Alzheimer's
Disease>. In addition, "suppressing" of an activity or
expression of a kinase protein and related terms mean to include
not only complete suppression (inhibition) but also partial
suppression of the activity or expression.
[0219] The "compound capable of suppressing the expression or
activity of the kinase protein" is not particularly limited, and
may be a known compound or may be one identified by the screening
to be described later. Examples of such a compound include
low-molecular-weight compounds capable of binding to the kinase
protein, RNAs capable of binding to a transcription product of a
gene encoding the kinase protein, antibodies against the kinase
protein, and peptides having a dominant negative phenotype against
the kinase protein.
[0220] Examples of the low-molecular-weight compound for PKC
include PKC inhibitors such as Go6976, UCN-01, BAY43-9006,
RO318220, RO320432, Isis3521, LY333531, LY379196,
bisindolylmaleimide, sphingosine, staurosporine, midosutaurin,
tyrphostin 51, hypericin, enzastaurin, rottlerin, safingol,
bryostatin 1, perifosine, and llmofosine. Examples thereof for CaMK
include CaMK inhibitors such as KN-93, KN-62, AIP, CaM kinase II
inhibitor 281-301, lavendustin C, K252a, rottlerin, ML-7, ML-9,
STO-609, W-7, and W-5. Examples thereof for CSK include CSK
inhibitors such as TBCA, IQA, TMCB, quinalizarin, quercetin, and
apigenin. Moreover, an example thereof for Lyn includes INNO-406
(NS-187). Examples thereof for b-RAF include b-raf inhibitors such
as PLX-4720 (N-[3-[(5-chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)carbony
l]-2,4-difluorophenyl]-1-propanesulfonamide), sorafenib
(4-[4-[3-[4-chloro-3-(trifluoromethyl)phenyl]ureido]phenoxy]-N-methylpyri-
dine-2-carboxamide), GDC-0879
(2-{4-[(1E)-1-(hydroxyimino)-2,3-dihydro-1H-inden-5-yl]-3-(pyridine-4-yl)-
-1H-pyrazol-1-yl}ethan-1-ol), vemurafenib (PLX4032, RG7204,
N-{3-[5-(4-chlorophenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluo-
rophenyl}propane-1-sulfonamide), dabrafenib
(N-[3-[5-(2-aminopyridin-4-yl)-2-tert-butyl-1,3-thiazol-4-yl]-2-fluorophe-
nyl]-2,6-difluorobenzenesulfonamide), sorafenib tosylate
(4-(4-{3-[4-chloro-3-(trifluoromethyl)phenyl]ureido}phenoxy)-N.sup.2-meth-
ylpyridine-2-carboxamide mono(4-methylbenzenesulfonate), and LGX818
(methyl[(2S)-1-{[4-(3-{5-chloro-2-fluoro-3-[(methylsulfonyl)amino]phenyl}-
-1-isopropyl-1H-pyrazol-4-yl)-2-pyrimidinyl]amino}-2-propanyl]carbamate).
[0221] In the present invention, examples of the "RNAs capable of
binding to a transcription product of a gene encoding a protein"
include dsRNAs (double-stranded RNAs), such as siRNAs and shRNAs
(short haipin RNAs), complementary to the transcription product of
the gene encoding the substrate protein or the kinase protein. The
length of such a dsRNA is not particularly limited, as long as the
expression of the target gene can be suppressed and no toxicity is
demonstrated. The length is, for example, 15 to 49 base pairs,
preferably 15 to 35 base pairs, and furthermore preferably 21 to 30
base pairs. The dsRNA does not necessarily have to have completely
the same base sequence as that of the target gene, but the homology
of the sequences is at least 70% or more, preferably 80% or more,
and furthermore preferably 90% or more (for example, 95%, 96%, 97%,
98%, 99% or more). The homology of the sequences can be determined
with a BLAST program.
[0222] Examples of other forms of the "RNAs capable of binding to a
transcription product of a gene encoding a protein" include
antisense RNAs complementary to the transcription product of the
gene encoding the substrate protein or the kinase protein; and RNAs
(ribozymes) having a ribozyme activity of specifically cleaving the
transcription product.
[0223] The above-described RNAs may have some or all of RNAs
substituted by an artificial nucleic acid such as PNA, LNA, or ENA.
Moreover, in order to express these RNAs in a target to which the
agent of the present invention is administered, each of the RNAs
may be in the form of an expression vector carrying a DNA encoding
the RNA. Additionally, those skilled in the art can prepare such
RNAs by chemical synthesis using a commercially-available
synthesizer or the like.
[0224] Examples of the "peptides having a dominant negative
phenotype against a protein" for the substrate protein include
polypeptides (for example, partial peptides and decoy peptides
containing a phosphorylated site of the substrate protein) which
compete with a kinase protein in binding to a binding site on a
substrate protein, and the like. Moreover, examples thereof for the
kinase protein include polypeptides (for example, partial peptides
containing a phosphorylated site of the kinase protein) which
competitively inhibit the activation of the kinase protein, and the
like.
[0225] <Alzheimer's Disease Therapeutic Agent 3>
[0226] As described above, it has been revealed that suppressing
the phosphorylations of the substrate proteins such as MARCKS
successfully suppresses the pathology in the Alzheimer's disease
model mice. Thus, the pathology can also be suppressed by
suppressing bindings, which are required for the phosphorylations,
between the substrate proteins such as MARCKS and the kinase
proteins such as PKC.
[0227] Based on such findings, the present invention also provides,
as a third embodiment of the therapeutic agent against Alzheimer's
disease, an agent comprising a compound capable of suppressing a
binding of at least one substrate protein selected from the group
consisting of MARCKS, Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1,
SYT1, G3P, HS90A, CLH, NFH, NFL, GPRIN1, ACON, ATPA, and ATPB to at
least one kinase protein selected from the group consisting of PKC,
CaMK, CSK, Lyn, and b-RAF.
[0228] The substrate proteins such as MARCKS and the kinase
proteins such as PKC targeted by the therapeutic agent of the
present invention are as described above in the description of
<Diagnostic Agent against Alzheimer's Disease>. Nevertheless,
the substrate proteins are preferably not phosphorylated, while the
kinase proteins are preferably activated kinase proteins. In
addition, "suppressing" of a binding between these proteins and
related terms mean to include not only complete suppression
(inhibition) but also partial suppression of the binding.
[0229] The "compound capable of suppressing the binding" is not
particularly limited, and may be a known compound or may be one
identified by the screening to be described later. Examples of such
a compound include polypeptides, antibodies, and
low-molecular-weight compounds all of which compete with the
substrate proteins and the kinase proteins in binding to a binding
site on the substrate proteins or the kinase proteins. Note that
the low-molecular-weight compounds of the present invention also
include physiologically acceptable salt or solvate forms of the
low-molecular-weight compounds.
[0230] <Alzheimer's Disease Therapeutic Agent 4>
[0231] As will be described later in Examples, it has been revealed
that activating Lyn also successfully suppresses the pathology in
the Alzheimer's disease model mice. Thus, the present invention
also provides, as a therapeutic agent against Alzheimer's disease,
an agent comprising a compound capable of activating Lyn. Lyn
targeted by the therapeutic agent of the present invention is as
described above in the description of <Diagnostic Agent against
Alzheimer's Disease>.
[0232] The "compound capable of activating Lyn" is not particularly
limited, and may be a known compound. Examples of such a compound
include low-molecular-weight compounds capable of binding to Lyn.
More concretely, examples thereof include Lyn kinase activators
described in International Publication No. WO2008/103692 (MLR-1023
and the like).
[0233] In addition, increasing an amount of Lyn expressed can also
increase the activity of Lyn. Thus, the "compound capable of
activating Lyn" also includes: nucleic acids (DNA, RNA) encoding
Lyn; DNA constructs (for example, plasmid DNA, viral vector)
capable of expressing Lyn, which is encoded by the nucleic acids,
in target cells; and Lyn proteins.
[0234] Hereinabove, preferred embodiments of the therapeutic agent
of the present invention have been described. In addition, the
therapeutic agent of the present invention may comprise, besides
the above-described compounds, the aforementioned other
pharmacologically acceptable ingredients, as in the case of the
diagnostic agents described above. Further, the therapeutic agent
of the present invention may also comprise a carrier for
introducing a nucleic acid, a protein, or the like into cells.
Examples of the carrier include substances having a positive charge
such as cationic liposome, and lipophilic substances (cholesterols
and derivatives thereof, lipids (such as, for example, glycolipids,
phospholipids, sphingolipids), vitamins such as vitamin E
(tocopherols)). Additionally, the therapeutic agent of the present
invention may be used in combination with known pharmaceutical
drugs which are used in the treatment of Alzheimer's disease.
[0235] The mode of administering the therapeutic agent of the
present invention is not particularly limited, and examples thereof
include intravenous administration, intraarterial administration,
intraperitoneal administration, subcutaneous administration,
intradermal administration, tracheobronchial administration, rectal
administration and intramuscular administration, administration by
transfusion, and direct administration into a target site (such as
brain). From the viewpoints that the therapeutic effect is high and
that an amount of the agent to be administered is small, the direct
administration into a target site is preferable. The administration
to a target site can be achieved by employing, for example, cannula
(catheter), surgical incision, drug delivery system, injection, or
the like. More concretely, examples thereof include a method in
which a cannula or the like is inserted by stereotactic surgery to
administer the agent into the brain through the cannula; a method
in which after a craniotomy, a sustained-release drug delivery
system (for example, ALZET osmotic pump) with the agent is
implanted into the brain; and a method in which the agent is
introduced into cells in the brain by electropolation. Meanwhile,
in the case where the agent of the present invention is not
directly administered into the brain, it is possible to utilize a
method in which a brain barrier-permeable substance is bound to the
compound and administered. Note that an example of the brain
barrier-permeable substance includes a 29-amino-acid glycoprotein
derived from rabies virus (see Nature, 2007 Jul. 5, Vol. 448, pp.
39 to 43), but is not limited thereto.
[0236] An amount of the therapeutic agent of the present invention
administered and the number of administrations can be adjusted as
appropriate depending on the type of the compounds, the body weight
and symptom of a test subject, and the like. The number of
administrations can be adjusted as appropriate depending on the
administration amount, the administration route, and the like.
[0237] A product of the therapeutic agent of the present invention
or a manual thereof may be provided with an indication stating that
the product is used for treating Alzheimer's disease. "A product or
a manual provided with an indication" is as described above in the
description of <Diagnostic Agent against Alzheimer's
Disease>. The indication may include information on an action
mechanism of the agent of the present invention such as information
that administering the agent of the present invention suppresses
phosphorylations of the substrate proteins such as MARCKS, thereby
suppressing abnormal spine formation or the like, and alleviating a
pathology of Alzheimer's disease.
[0238] Moreover, the present invention also makes it possible to
treat Alzheimer's disease by administering the compound to a
subject as described above. Thus, the present invention also
provides a method for treating Alzheimer's disease, the method
characteristized by comprising administering to a subject any one
of: a compound capable of suppressing a phosphorylation of at least
one substrate protein selected from the group consisting of MARCKS,
Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A, CLH,
NFH, NFL, GPRIN1, ACON, ATPA, and ATPB; a compound capable of
suppressing an activity or expression of at least one kinase
protein selected from the group consisting of PKC, CaMK, CSK, Lyn,
and b-RAF; a compound capable of suppressing a binding of at least
one substrate protein selected from the group consisting of MARCKS,
Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A, CLH,
NFH, NFL, GPRIN1, ACON, ATPA, and ATPB to at least one kinase
protein selected from the group consisting of PKC, CaMK, CSK, Lyn,
and b-RAF; and a compound capable of activating Lyn.
[0239] <Screening Method 1 for Alzheimer's Disease Therapeutic
Agent Candidate Compound>
[0240] As described above, it has been found that suppressing the
expressions of the substrate proteins such as MARCKS in the
Alzheimer's disease model mice suppresses the phosphorylations of
the protein, thereby successfully suppressing the pathology in the
mice. Thus, based on such findings, the present invention also
provides the following screening method for a candidate compound
for treating Alzheimer's disease, the method comprising: [0241] (i)
a step of applying a test compound to a system capable of detecting
a phosphorylation of at least one substrate protein selected from
the group consisting of MARCKS, Marcksl1, SRRM2, SPTA2, ADDB, NEUM,
BASP1, SYT1, G3P, HS90A, CLH, NFH, NFL, GPRIN1, ACON, ATPA, and
ATPB; and [0242] (ii) a step of selecting the compound if the
compound suppresses the phosphorylation of the substrate
protein.
[0243] The "substrate proteins such as MARCKS" used in this
screening method are as described above in the description of
<Screening Method for Alzheimer's Disease Diagnostic Agent
Candidate Compound>. These proteins may be partial peptides, but
need to contain at least a phosphorylated site(s). Moreover, other
proteins such as a tag protein for purification may be added to
these proteins.
[0244] Further, the "test compound" used in this screening method
is not particularly limited. Examples thereof include the compounds
described above in <Screening Method for Alzheimer's Disease
Diagnostic Agent Candidate Compound>.
[0245] The "system capable of detecting the phosphorylation of the
substrate protein" is not particularly limited. An example thereof
includes a mixture solution of the substrate proteins such as
MARCKS and the kinase proteins (such as PKC) which phosphorylate
the substrate proteins. Moreover, a radiolabeled phosphate is added
together with a test compound to this mixture solution, followed by
incubation to detect the incorporation of the phosphate into the
substrate protein with a scintillation counter, by autoradiography,
or other means. Then, if an amount of the phosphate incorporated
into the substrate protein detected in the presence of the test
compound is small in comparison with that detected in the absence
of the test compound, the test compound is evaluated as a compound
which suppresses the phosphorylation of the substrate protein, and
selected as a candidate compound for treating Alzheimer's
disease.
[0246] Additionally, an example of another embodiment of the
"system capable of detecting the phosphorylation of the substrate
protein" includes a system capable of directly detecting the
phosphorylation of the substrate protein such as MARCKS. Regarding
this system, a test compound is applied to cells expressing the
substrate protein or cells in which the protein is forcibly
expressed, and the phosphorylation of the substrate protein in the
cells is detected. Then, if an amount of the phosphorylated
substrate protein detected is small in comparison with that
detected in the absence of the test compound, the test compound is
evaluated as a compound which suppresses the phosphorylation of the
substrate protein. In the case where the phosphorylation of the
substrate protein is directly detected, it is suitable to employ a
detection method using an antibody capable of specifically binding
to a phosphorylation site of a substrate protein, a method
utilizing a detector based on the surface plasmon resonance
phenomenon using a thin metal film on which a compound capable of
specifically binding to a phosphorylation site of a substrate
protein is immobilized, and the like as in <Method 1 for
Diagnosing Alzheimer's Disease> described above.
[0247] <Screening Method 2 for Alzheimer's Disease Therapeutic
Agent Candidate Compound>
[0248] As described above, it has been found that suppressing
activations of the kinase proteins such as PKC in the Alzheimer's
disease model mice also successfully suppresses the pathology in
the mice. Thus, based on such a finding, the present invention also
provides the following method as a second embodiment of the
screening method for a candidate compound for treating Alzheimer's
disease, the method comprising: [0249] (i) a step of applying a
test compound to a system capable of detecting an activity or
expression of at least one kinase protein selected from the group
consisting of PKC, CaMK, CSK, Lyn, and b-RAF; and [0250] (ii) a
step of selecting the compound if the compound suppresses the
activity or expression of the protein.
[0251] As in <Method for Diagnosing Alzheimer's Disease>
described above, the "kinase proteins such as PKC" used in this
screening method are not limited respectively to the proteins
having the amino acid sequences listed as the typical examples
described above, and naturally-occurring mutants thereof can also
be targeted.
[0252] The "system capable of detecting the activity of the kinase
protein such as PKC" should be a system capable of detecting the
activity of the kinase protein, that is, the phosphorylation of the
substrate protein. It is suitable to use the systems described
above in <Screening Method 1 for Alzheimer's Disease Therapeutic
Agent Candidate Compound>. Moreover, since a phosphorylation of
a substrate protein requires the activation (such as
phosphorylation) of a kinase protein per se, a "system capable of
detecting a phosphorylation of a kinase protein such as PKC" may
also be used. Note that this system is constructed using the kinase
proteins such as PKC in place of the substrate proteins such as
MARCKS in the "system capable of detecting the phosphorylation of
the substrate protein" described above.
[0253] Examples of the "system capable of detecting the expression
of the kinase protein such as PKC" include cells having a DNA in
which a reporter gene is operably linked downstream of a promoter
region of a gene encoding the each kinase protein, or liquid
extracts from the cells. Herein, the phrase "operably linked"
refers to linking of the reporter gene to the promoter region of
each gene such that the expression of the reporter gene is induced
by binding of a transcription factor to the promoter region of the
gene. Moreover, a test compound is applied to this system to
measure an activity of a protein encoded by the reporter gene. If
the detected activity is low in comparison with that detected in
the absence of the test compound, the test compound is evaluated as
having an activity of suppressing the expression of each kinase
protein.
[0254] An example of another embodiment of the "system capable of
detecting the expression of the kinase protein such as PKC" besides
the above-described reporter system includes a system capable of
directly detecting the expression of the kinase protein such as
PKC. Regarding this system, a test compound is applied to cells
expressing each protein, and the expression of each protein in the
cells is detected. Then, if an detected amount of each protein
expressed is small in comparison with that detected in the absence
of the test compound, the test compound is evaluated as having an
activity of suppressing the expression of the protein. In detecting
the expression of the protein, in the case where the expression of
the protein per se is to be detected, it is suitable to employ a
detection method using an antibody capable of specifically binding
to a kinase protein (preferably, activated kinase protein), a
method utilizing a detector based on the surface plasmon resonance
phenomenon using a thin metal film on which a compound capable of
specifically binding to a kinase protein (preferably, activated
kinase protein) is immobilized, and the like as in <Method 2 for
Diagnosing Alzheimer's Disease> described above. Meanwhile, in a
case of detecting the expression of the kinase protein through the
gene expression at a transcription level, a northern blotting
method, an RT-PCR method, a dot blotting method, or the like can be
employed.
[0255] <Screening Method 3 for Alzheimer's Disease Therapeutic
Agent Candidate Compound>
[0256] As described above, it has been revealed that suppressing
the phosphorylations of the substrate proteins such as MARCKS
successfully suppresses the pathology in the Alzheimer's disease
model mice. Thus, the pathology can also be suppressed by
suppressing bindings, which are required for the phosphorylations,
between the substrate proteins such as MARCKS and the kinase
proteins such as PKC.
[0257] Based on such findings, the present invention also provides
the following method as a third embodiment of the screening method
for a candidate compound for treating Alzheimer's disease.
[0258] A screening method for a candidate compound for treating
Alzheimer's disease, the method comprising the following steps (a)
to (c): [0259] (a) a step of bringing at least one kinase protein
selected from the group consisting of PKC, CaMK, CSK, Lyn, and
b-RAF into contact with at least one substrate protein selected
from the group consisting of MARCKS, Marcksl1, SRRM2, SPTA2, ADDB,
NEUM, BASP1, SYT1, G3P, HS90A, CLH, NFH, NFL, GPRIN1, ACON, ATPA,
and ATPB, in presence of a test compound; [0260] (b) a step of
detecting a binding between the kinase protein and the substrate
protein; and [0261] (c) a step of selecting the compound if the
compound suppresses the binding.
[0262] The "kinase proteins such as PKC," the "substrate proteins
such as MARCKS," and the "test compound" used in this screening
method are as described above in the description of <Screening
Method for Alzheimer's Disease Diagnostic Agent Candidate
Compound>. Nevertheless, the substrate proteins are preferably
not phosphorylated, while the kinase proteins are preferably
activated kinase proteins.
[0263] In the step (a), the kinase protein and the substrate
protein are brought into contact with each other in the presence of
the test compound. The contact should be conducted under conditions
that would not inhibit the binding in the absence of the test
compound.
[0264] In the step (b), the binding between the kinase protein and
the substrate protein is detected. This binding detection is not
particularly limited, and a known method can be employed as
appropriate. For example, it is possible to employ a co-immuno
precipitation method, an ELISA method, a method using a detector
based on the surface plasmon resonance phenomenon, or a method
based on FRET.
[0265] In the step (c), the compound is selected if the compound
suppresses the binding. For example, when the
co-immunoprecipitation method is employed, the evaluation is
possible by comparison between an amount of the substrate protein
coprecipirated when the kinase protein is precipitated by an
antibody specific thereto in the presence of the test compound and
an amount (control value) of the substrate protein in the absence
of the test compound. To be more specific, if the amount of the
substrate protein in the presence of the test compound is small in
comparison with the amount in the absence of the test compound, the
test compound can be evaluated as a candidate compound for treating
Alzheimer's disease. When a method other than the
immunoprecipitation method is employed in the detection of the
binding, a similar evaluation is possible by using the degree of
the binding in the absence of the test compound as a control
value.
[0266] Hereinabove, preferred embodiments of the screening method
for a candidate compound for treating Alzheimer's disease of the
present invention have been described. In addition, in the
screening method of the present invention, it is possible to
further narrow down a candidate compound on the basis of a recovery
from a symptom of an Alzheimer's disease model animal in which the
compound selected according to the above-described methods has been
administered.
[0267] Examples of the "Alzheimer's disease model animals" include,
as described later in Examples, animals (such as mice, rats,
marmosets) in which an Alzheimer's disease responsible gene (such
as PS1 exon 9 deletion mutant; PS2 mutant (N141I); human
double-mutant APP695 (KM670/671NL, Swedishtype); quintuple mutant
(human APP695 triple mutant with Swedish type (KM670/671NL),
Florida type (I716V), and London type (V717I), as well as human PS1
double mutant (M146L and L285V)); human tau mutant; or a
combination of these mutants) is introduced.
[0268] The "recovery from a symptom of a model animal" can be
detected, for example, as described later in Examples, by
performing in vivo imaging with a two-photon microscope on the
degree of a recovery from abnormal spine formation caused by
Alzheimer's disease. Moreover, the detection is also possible by
conducting a behavioral test described later in Examples and
evaluating the degree of a recovery from an abnormal behavior of
the model animal.
[0269] Hereinabove, preferred embodiments of the diagnosis method,
the diagnostic agent, and the therapeutic agent against Alzheimer's
disease, the screening methods for candidate compounds of these
agents of the present invention, and so forth have been described.
Hereinafter, description will be given of a diagnostic agent, a
therapeutic agent, and so forth against frontotemporal lobar
degeneration.
[0270] <Method for Diagnosing Frontotemporal Lobar
Degeneration>
[0271] As will be described later in Examples, it has been revealed
that a b-RAF protein which is a kinase protein phosphorylating
substrate proteins such as tau protein is activated in
frontotemporal lobar degeneration model mice before the onset of
the disease. Thus, the present invention also provides a method for
diagnosing frontotemporal lobar degeneration, the method comprising
the following the steps (i) to (iii): [0272] (i) a step of
detecting an activity or expression of a b-RAF protein in a test
subject; [0273] (ii) a step of comparing the activity or expression
with an activity or expression of a b-RAF protein in a normal
subject; and [0274] (iii) a step of determining that the test
subject is affected with frontotemporal lobar degeneration or has a
risk of developing frontotemporal lobar degeneration if the
activity or expression of the b-RAF protein in the test subject is
higher than the activity or expression of the b-RAF protein in the
normal subject as a result of the comparison.
[0275] This diagnosis method is a method similar to <Method for
Diagnosing Alzheimer's Disease> described above. In addition,
examples of the "activated kinase protein" to be detected include,
as described above, b-RAF having serine at position 365
phosphorylated, b-RAF having serine at position 446 phosphorylated,
b-RAF having serine at position 579 phosphorylated, b-RAF having
threonine at position 599 phosphorylated, b-RAF having serine at
position 602 phosphorylated, b-RAF having serine at position 729
phosphorylated, and b-RAF having serine at position 732
phosphorylated. From the viewpoint of having a larger difference
between a subject affected with frontotemporal lobar degeneration
or having a risk of developing and a normal subject, the detection
target in the method for diagnosing frontotemporal lobar
degeneration of the present invention is preferably b-RAF having
serine at position 365 phosphorylated, b-RAF having serine at
position 729 phosphorylated, and b-RAF having serine at position
732 phosphorylated, and the detection target is more preferably
b-RAF having serine at position 729 phosphorylated.
[0276] Moreover, the present invention makes it possible to
determine that one is affected with frontotemporal lobar
degeneration or has a risk of developing frontotemporal lobar
degeneration. In this manner, enabling judgment of frontotemporal
lobar degeneration affection or the like at an early stage leads to
an expection that treatment methods for suppressing a pathology of
frontotemporal lobar degeneration (such a method for administering
an agent for suppressing a pathology of frontotemporal lobar
degeneration) will be effective.
[0277] Thus, the present invention also makes it possible to
provide, as in the case of <Method for Diagnosing Alzheimer's
Disease> described above, a method for treating frontotemporal
lobar degeneration, the method comprising a step of administering
an agent for suppressing a pathology of frontotemporal lobar
degeneration to a test subject determined to be affected with
frontotemporal lobar degeneration or have a risk of developing
frontotemporal lobar degeneration by the diagnosis method of the
present invention.
[0278] Additionally, examples of the agent administered to suppress
a pathology of frontotemporal lobar degeneration include
serotonin-specific reuptate inhibitors (SSRI), cholinesterase
inhibitors (ChEI), agents for suppressing tau aggregation (such as
aminothienopyridazine, cyanine dye, methylene blue), and
neuroprotective drugs (such as dimebon).
[0279] Further, as will be described later, it is also possible to
suitably use, as the agent for suppressing a pathology of
frontotemporal lobar degeneration, an agent for treating
frontotemporal lobar degeneration, the agent comprising a compound
capable of suppressing an activity or expression of a b-RAF
protein.
[0280] <Diagnostic Agent Against Frontotemporal Lobar
Degeneration>
[0281] As described above, it has been revealed that the kinase
protein b-RAF which phosphorylates the substrate proteins such as
tau protein is activated before the onset of the disease. Thus, the
present invention provides an agent for diagnosing frontotemporal
lobar degeneration, the agent comprising a compound having an
activity of binding to b-RAF.
[0282] As in the case of <Method for Diagnosing Alzheimer's
Disease> described above, the b-RAF protein targeted by the
diagnostic agent against frontotemporal lobar degeneration of the
present invention is not limited to the proteins having the amino
acid sequences listed as the typical examples described above, and
naturally-occurring mutants can also be targeted. Moreover, the
"compound having an activity of binding to b-RAF" is not
particularly limited, and it is possible to similarly use the
compounds described above in <Diagnostic Agent against
Alzheimer's Disease>.
[0283] <Therapeutic Agent Against Frontotemporal Lobar
Degeneration>
[0284] As will be described later in Examples, it has also been
revealed that the b-RAF protein which phosphorylates the substrate
proteins such as tau protein is activated in the frontotemporal
lobar degeneration model mice before the onset of the disease.
Further, it has also been found that suppressing the activation of
the b-RAF protein by using an inhibitor against the protein
successfully suppresses the pathology in the frontotemporal lobar
degeneration model mice.
[0285] Thus, the present invention also provides, as a therapeutic
agent against frontotemporal lobar degeneration, an agent
comprising a compound capable of suppressing an activity or
expression of b-RAF.
[0286] The frontotemporal lobar degeneration therapeutic agent of
the present invention is similar to <Alzheimer's disease
therapeutic agent> described above. Moreover, the targeted b-RAF
protein is as described above in the description of <Diagnostic
Agent against Frontotemporal Lobar Degeneration>.
[0287] As in the case of <Alzheimer's disease therapeutic
agent> described above, a product of the frontotemporal lobar
degeneration therapeutic agent of the present invention or a manual
thereof may be provided with an indication stating that the
therapeutic agent is used for treating frontotemporal lobar
degeneration. "A product or a manual provided with an indication"
is as described above in the description of <Diagnostic Agent
against Alzheimer's Disease>. The indication may include
information on an action mechanism of the agent of the present
invention such as information that administering the agent of the
present invention suppresses b-RAF activity, thereby suppressing a
decrease in the number of spines and so forth, and alleviating a
pathology of frontotemporal lobar degeneration.
[0288] Moreover, the present invention also makes it possible to
treat frontotemporal lobar degeneration by administering the
compound to a subject as described above. Thus, the present
invention also provides a method for treating frontotemporal lobar
degeneration, the method characterized by comprising administering
to a subject a compound capable of suppressing an activity or
expression of b-RAF.
EXAMPLES
[0289] Hereinafter, the present invention will be more specifically
described on the basis of Examples. However, the present invention
is not limited to the following Examples.
[0290] --Alzheimer's Disease--
[0291] In the present Examples, first, experimental methods and so
forth described below were carried out to identify phosphoproteins
and kinase proteins which played central roles in a pre-onset stage
of Alzheimer's disease, as well as a network composed of these
proteins, and consequently to provide target molecules useful in
the diagnosis and treatment of Alzheimer's disease.
[0292] <Experiments Using Model Mice>
[0293] The following five types of Alzheimer's disease model mice
were used in the present Examples.
(1) PS1 transgenic mice (mice expressing exon 9 deletion mutant
(PSENIdE9) under the control of the mouse PrP promoter; see
Jankowsky, J. L. et al., Hum. Mol. Genet., 2004, Vol. 13, pp. 159
to 170) (2) PS2 transgenic mice (mice expressing human PS2 mutant
(N141I) under the control of the ubiquitous CMV early enhancer and
the chicken .beta. actin promoter; see Oyama, F. et al., J.
Neurochem., 1998, Vol. 71, pp. 313 to 322) (3) Human double-mutant
APP695 (KM670/671NL, Swedishtype) transgenic mice (the mice were
prepared by substituting a mutant for the PrP gene in a hamster PrP
cosmid vector (see Hsiao, K. et al., Science, 1996, Vol. 274, pp.
99 to 102)) (4) 5.times.FAD mice (transgenic mice expressing human
APP695 having Swedish type (KM670/671NL), Florida type (I716V), and
London type (V717I) triple mutations, as well as human PS1 having
double mutations (M146L and L285V) under the control of mouse Thy1)
(see Oakley, H. et al., J. Neurosci., 2006, Vol. 26, pp. 10129 to
10140) (5) Transgenic mice expressing a human tau mutant protein
under the control of the mouse PrP promoter (see Yoshiyama, Y. et
al., Neuron, 2007, Vol. 53, pp. 337 to 351).
[0294] Note that the genetic backgrounds of the transgenic mice
were C57BL/6J, C57BL/6J, C57/B6XSJL, C57/B6XSJL, and B6C3H/F1,
respectively.
[0295] In the mass spectrometry to be described later, brain
tissues were isolated from male transgenic mice described above at
the age in months shown in figures and descriptions thereof, and
subjected to the analysis.
[0296] In the immunohistochemical analysis, the brain samples were
fixed with 4% paraformaldehyde, and paraffin sections were prepared
(the thickness of each section: 5 .mu.m) using a microtome
(manufactured by Yamato Kohki Industrial Co., Ltd.). Meanwhile, the
following antibodies were each diluted to 1/1000 and used as
primary antibodies.
Anti-A.beta. antibody (82E1), manufactured by IBL Co., Ltd., Code
No: 10323 Anti-A.beta. antibody (6E10), manufactured by Covance
Inc., Product Code: SIG-39300 Anti-human PHF-tau antibody (AT-8),
manufactured by Innogenetics N.V., Catalog No: BR-03.
[0297] Then, the tissue samples reacted with each antibody were
treated with VECTASTAIN Elite ABC Kit and DAB Peroxidase Substrate
Kit (manufactured by Vector Laboratories) to visualize the
expressions of proteins recognized by the antibodies.
[0298] Moreover, although unillustrated, male transgenic mice
described above were subjected to six behavioral tests described
below. Based on detected abnormal behaviors, whether or not these
mice developed Alzheimer's disease was evaluated.
(1) Morris Water Maze Test
[0299] In this test, the mice received a 60-second trial four times
a day for 5 days. The time until each mouse reached the platform
was measured.
(2) Rotarod Test
[0300] In this test, a trial was conducted four times a day for 3
days in which a mouse was allowed to grab on a rotating rod
(rotation speed: 3.5 to 35 rpm) with the speed being gradually
increased. The average time until the mouse fell from the rotating
rod was recorded.
(3) Fear-Conditioning Test
[0301] In this test, first, a mouse received a sound stimulus
(65-dB white noise, 30 seconds) together with an electrical
stimulus (0.4 mA, 2 seconds) on the foot. Then, after 24 hours, the
mouse was measured for the frequency of the freezing reaction when
the mouse received a sound stimulus but no electrical stimulus in
the same chamber.
(4) Open-Field Test
[0302] In this test, the time during which a mouse stayed in a
central region of an open field was measured.
(5) Light-Dark Box Test
[0303] In this test, the time during which a mouse stayed in a
light box was measured.
(6) Elevated Plus Maze Test
[0304] In this test, the time during which a mouse stayed on arms
with no walls in an elevated plus maze set 60 cm above the floor
was measured.
[0305] <Human Brain>
[0306] For a proteome analysis to be described later, brain samples
were isolated from AD (Alzheimer's disease) patients, DLB (dementia
with lewy bodies) patient, and healthy control persons and frozen
at -80.degree. C. within 1 hour after death. Moreover, temporal
pole and occipital pole tissues were dissected from five brains in
each group.
[0307] Note that a neuropathologist pathologically diagnosed each
brain sample based on the immunohistochemistry. As a result, in the
brains of the AD patients, other pathologies such as lewy bodies,
TDP43 cytoplasmic aggregates, and argyrophilic grains were not
observed. Moreover, in the brains of the DLB patients, the
disease-specific pathological observation was confirmed.
[0308] <Preparation of Phosphoproteins and
Phosphopeptides>
[0309] In preparing phosphoproteins and phosphopeptides from the
transgenic mice and so forth, first, mice were euthanized using
ethyle ether. Within 5 minutes thereafter, the cerebral cortexes
were collected. The obtained cerebral cortexes were immediately
frozen with liquid nitrogen and stored until phosphoproteins were
extracted. In the protein extraction, first, the cortical tissues
were lysed with a cold lysis buffer containing 2% SDS, 1 mM DTT,
and 100 mM Tris-HCl (pH 7.5). The cells were disrupted with 20
strokes of a glass Dounce homogenizer on ice. The ratio of the
lysis buffer to the tissue was 10 .mu.L to 1 mg. After the cells
were disrupted, the lysate was incubated at 100.degree. C. for 15
minutes. Then, the crude extract was obtained by centrifugation at
4.degree. C. at 16000.times.g for 10 minutes. The collected
supernant was diluted to a 1/10 concentration with water, and
filtered through a filter having a pore diameter of 0.22 .mu.m. The
resulting flow-through fraction was concentrated to a 10-fold
concentration using an Amicon Ultra3K filter (manufactured by
Millipore Corporation). Further, the concentrations of proteins
thus prepared were measured using the BCA Protein Assay Reagent
(manufactured by Thermo Fisher Scientific Inc.).
[0310] Subsequently, a solution of 100 .mu.L of 1 M
triethylammonium bicarbonate (TEAB) (pH 8.5), 3 .mu.L of 10% SDS,
and 30 .mu.L of 50 mM tris-2-carboxyethyl phosphine (TCEP) was
added to sample aliquots (200 .mu.L) containing 15 mg of the
proteins, and incubated at 60.degree. C. for 1 hour. Moreover, to
protect cysteine residues, 10 mM methyl methanethiosulfonate (MMTS)
was added and treated at 25.degree. C. for 10 minutes. Thereafter,
the obtained sample was treated at 37.degree. C. for 24 hours with
80 mM CaCl.sub.2 and trypsin (mass spectrometry grade) (10:1
protein/enzyme, w/w). Then, phosphopeptides were concentrated using
TITANSPHERE.RTM. Phos-Tio Kit (manufactured by GL Sciences Inc.)
according to the instruction, and desalted using a Sep-Pak Light
C18 cartridge column (manufactured by Waters Corporation) according
to the instruction. The sample aliquots were dried and then
dissolved in 25 .mu.L of 100 mM TEAB (pH 8.5). Further, the
phosphopeptide in each sample were labeled separately using the
iTRAQ.RTM. multiplex assay kit (manufactured by AB SCIEX Ins.) at
25.degree. C. for 2 hours according to the instruction.
Subsequently, the labeled phosphopeptide pools were mixed together.
The obtained aliquots were dried and then re-dissolved in 1 mL of
0.1% formic acid.
[0311] <2D LC MS/MS Analysis>
[0312] The phosphopeptide samples labeled as described above were
subjected to strong cation exchange (SCX) chromatography using a
TSK gel SP-5PW column (manufactured by TOSHO Corporation) and a
Prominence UFLC system (manufactured by Shimadzu Corporation). Note
that the flow rate was 10 mL/minute with solution A (10 mM
KH.sub.2PO.sub.4 (pH 3.0), 25% acetonitrile). Thereafter, elution
was performed using solution B (10 mM KH.sub.2PO.sub.4 (pH 3.0),
25% acetonitrile, 1 M KCl) in a gradient range of 0 to 50%. The
collected elution fractions were dried and then re-dissolved in 100
.mu.L of 0.1% formic acid.
[0313] Subsequently, each fraction thus prepared was analyzed using
a DiNa Nano-Flow LC system (manufactured by KYA Technologies
Corporation) and Triple TOF 5600 System (manufactured by AB SCIEX
Ins.). In the liquid chromatography, samples were loaded onto a 0.1
mm.times.100 mm C18 column together with solution C (2%
acetonitrile and 0.1% formic acid) and eluted using solution D (80%
acetonitrile and 0.1% formic acid) in a gradient range of 0 to 50%.
Note that the flow rate was set at 300 nL/minute, and the ion spray
voltage was set at 2.3 kV. The information-dependent acquisition
(IDA) was performed in a range of 400 to 1250 m/z with 2 to 5
charges. Moreover, to identify each peptide, the Analyst TF1.5
software (manufactured by AB SCIEX Ins.) was used. Further, each
peptide was quantified based on the TOF-MS current detected during
the LC-separated peptide peak, and adjusted to the charge/peptide
ratio. In addition, the obtained signals were analyzed using
Analyst TF (version 1.5) (manufactured by AB SCIEX Ins.). Then, the
signals were processed by ProteinPilot software (version 4).
[0314] <Data Analysis>
[0315] As described above, in the 2D LC MS/MS analysis, the mass
spectra of the peptides were acquired and analyzed using Analyst TF
(version 1.5) (manufactured by AB SCIEX Ins.). Then, based on the
obtained result, corresponding proteins were searched using human
and mouse protein sequence database (UniProtKB/Swiss-Prot, data
downloaded from UniProt (http://www.uniprot.org) on 2010 Jun. 22,
with ProteinPilot software (version 4) including Paragon algorithm
(manufactured by AB SCIEX Ins., see Shilov, I. V. et al., Mol.
Cell. Proteomics, 2007, Vol. 6, pp. 1638 to 1655) as described
above. Note that the tolerance for the searched of the peptides by
ProteinPilot was set to 0.05 Da for the MS analysis and 0.10 Da for
the MS/MS analysis. Moreover, in ProteinPilot, "phosphorylation
emphasis" was set at the sample description, and "biological
modifications" was set at the processing specification. Further,
the confidence score was utilized to evaluate the quality of the
peptide identification. Furthermore, the identified proteins were
grouped by the ProGroup algorithm (manufactured by AB SCIEX Ins.)
to exclude redundancy. Additionally, the threshold value for the
protein detection was set at 95% confidence in ProteinPilot. Then,
if the confidence was 95% or more, the protein was determined to be
identified.
[0316] Moreover, an MS/MS spectrum was prepared upon a
fragmentation in the mass spectrometer. Further, the proteins were
quantified through iTRAQ reporter group analysis in the MS/MS
spectrum. In the peptide and protein quantification, bias
correction option was used to normalize signals of different iTRAQ
reporters. In addition, peptide ratios, that is, ratios between
reporter signals in the AD patients and those in control samples,
were calculated after the bias correction. A protein ratio (average
ratio) was deduced from a weighted average of peptide ratios
corresponding to proteins. Moreover, the deduction used peptide
ratios differently weighted based on error factors after the bias
correction. Note that detailed formulas used to calculate these
values were described in the manual from ABSCIEX. Further, using
the peptide ratios, amounts of the proteins in the AD patients were
compared with those in the control samples. Student's t-value was
calculated from weighted average of log peptide ratio, its standard
error, and log bias. Furthermore, P-value was calculated together
with a post hoc test in ProteinPilot to exclude multiple hypothesis
testing-related problem. The P-values of three samples obtained in
this test were integrated by inverse normal method. Then, if the
integrated P-value was smaller than 0.05, it was determined that
the phosphorylations of the proteins were changed.
[0317] The peptide summary and protein summary in ProteinPilot were
inputted into Excel for further data analyses. Moreover, a
geometric mean of signal intensities derived from multiple MS/MS
fragments containing the phosphorylation site was calculated as an
amount of phosphopeptide fragment. Further, a difference between
the AD patient group and the control group was evaluated by
Student's t-test (n=3). Then, the changed phosphoproteins were
compared among different AD models, and proteins were selected
which commonly changed in a hypothesis free approach or an A.beta.
aggregation-linked approach.
[0318] <Systems Biology>
[0319] ProteinPilot software was used to identify proteins
expressed in the occipital lobe and the temporal lobe of the human
brain (see Shilov, I. V. et al., Mol. Cell. Proteomics, 2007, Vol.
6, pp. 1638 to 1655). To be more specific, ProteinPilot
automatically added Uniprot ID to each observed protein. Then, the
observed proteins were searched for proteins belonging to common
Homologene Group ID, and the Taxonomy IDs and Gene IDs of the
collected proteins were obtained. Note that the number of the
Taxonomy IDs was limited to 9606 for human, 10090 for mouse, and
10116 for rat. Moreover, Uniprot IDs of newly added proteins were
also attached. Next, from the list of the collected proteins,
proteins having Uniprot IDs not listed in the GNP database
(http://genomenetwork.nig.ac.jp/index_e.html) were excluded.
Subsequently, a database was created from information collected
from the GNP by utilizing a super computer system at the Human
Genome Center in the University of Tokyo. As a result, remaining
proteins were determined as analyzed proteins. Moreover, the GNP
database was searched for proteins linked to the analyzed proteins,
so that an edge file was created (redundant edges were excluded).
Based on the created edge file, a protein network was obtained and
visualized using Cell Illustrator (see Nagasaki, M. et al., Appl.
Bioinformatics, 2003, Vol. 2, pp. 181 to 184).
[0320] <In Vivo Imaging with Two-Photon Microscope>
[0321] Two-photon imaging of dendritic spine was performed using a
laser-scanning microscope system FV1000MPE2 (manufactured by
Olympus Corporation) equipped with an upright microscope (BX61WI,
manufactured by Olympus Corporation, a water-immersion objective
lens (XLPlanN25xW; numerical aperture, 1.05), and a pulsed laser
(MaiTai HP DeepSee, manufactured by Spectra Physics). In the
imaging, EGFP was excited by light at a wavelength of 890 nm, and
scanned in a range of 500 to 550 nm. Moreover, the scanned region
for three-dimensional imaging was 100.times.100 .mu.m (1 .mu.m
Z-axis steps, 1024.times.1024 pixels).
[0322] Additionally, two weeks before the imaging, adeno-associated
virus 1 (AAV1)-EGFP with the synapsin 1 promoter (titer:
1.times.10.sup.10 vector genomes/mL, 1 .mu.L) was injected into the
retrosplenial cortex (-2.0 mm anteroposterior and 0.6 mm
mediolateral from the bregma, depth 1 mm) of mice under anesthesia
with 2.5% isoflurane. Then, two weeks thereafter, the dendritic
spines of the first layer (layer 1) of the cerebral cortex were
observed through a thinned skull window according to the method
described in "Yang, G. et al., Nat. Protoc., 2010, Vol. 5, pp. 201
to 208."
[0323] Moreover, when the influence of kinase inhibition on
dendritic spines was imaged, Alzet micro-osmotic pumps (model:
1003D, manufactured by Durect Corporation) filled with PBS/1% DMSO
containing 1 .mu.M Go6976 (manufactured by Calbiochem), 0.4 mM
KN-93 (manufactured by Cayman Chemical), or 1 .mu.M MLR1023
(manufactured by Glixx Laboratories) were implanted into mice under
anesthesia with O.sub.2/isoflurane. Then, 30 hours or 60 hours
elapsed after the osmotic pumps were implanted, dendritic spines
were observed. Note that, regarding the PKC inhibitor Go6976, see
Yan, Z. et al., Proc. Natl. Acad. Sci. U.S.A, 1999, Vol. 96, pp.
11607 to 11612. Regarding the CaMK inhibitor KN-93, see Galan, A.
et al., Pain., 2004, Vol. 112, pp. 315 to 323. Regarding the Lyn
activator MLR1023, see Saporito, M. S. et al., J. Pharmacol. Exp.
Ther., 2012, Vol. 342, pp. 15 to 22.
[0324] Meanwhile, when the influence of shRNA-lentiviral vector
introduction on dendritic spines was imaged, 3 .mu.L of a
lentiviral vector encoding shRNA against MARCKS (sc-35858-V,
manufactured by Santa Cruz Biotechnology Inc., 1.times.10.sup.6 TU)
or scrambled shRNA (RHS4348, 1.times.10.sup.6 TU) was injected into
the same region as in the case of the AAV1-EGFP.
[0325] In addition, the spine density, spine length, spine maximum
diameter, and spine neck minimum diameter were measured from the
obtained images using image analysis software IMARIS 7.2.2
(manufactured by Bitplane).
[0326] <Statistical Analysis>
[0327] Mass spectrometry data on the disease model mice or human
patients were evaluated by inverse normal method in comparison with
data on the respective background mice or human control samples.
The amount of each peptide in the mass spectrometry was based on
multiple peaks, and the amount of each protein was based on
multiple peptide values. In consideration of these, P-values were
obtained for these amounts. Moreover, together with the P-values,
differential gene expression analysis was performed on each peptide
or protein without replication. Further, to guarantee the result
quality, biologically replicated data were also obtained which
could increase the number of identified proteins. As a result, it
was determined that appropriate sample sizes of human and mouse
brains were respectively N=5 and N=3.
[0328] Note that whether or not all the samples formed normal
distribution was not confirmed. Nevertheless, in the process of
calculating the P-value with a computer, low-quality measurement
results producing abnormal values were excluded in the present
analysis using the commercially-available program
(ProteinPilot).
[0329] Additionally, the results obtained by the animal behavioral
tests and two-photon microscope observation were basically analyzed
by Student's independent t-test (two-sided test) in the sample
sizes shown in figures and descriptions thereof.
[0330] Moreover, brain tissue sampling, data collection in the mass
spectrometry, and the systems biology analysis were performed by
independent researchers without assigning the tasks to groups who
knew the circumstances.
Example 1
[0331] <Phosphoproteome Analysis on Alzheimer's Disease>
[0332] It has been suggested that various phosphorylation signal
transductions including tau phosphorylation are involved in a
pathology of Alzheimer's disease. Accordingly, identifying
phosphorylation signal transductions in Alzheimer's disease,
particularly, a phosphorylation signal transduction which played a
central role in a pre-onset stage of Alzheimer's disease, makes it
possible to provide very effective target molecules in early-stage
diagnosis and treatment of this disease. Hence, the present
inventor made efforts to comprehensively analyze (phosphoproteome
analysis) phosphorylation signal transductions in Alzheimer's
disease to identify a phosphorylation signal transduction which
played a central roles in the pathology.
[0333] However, a postmortem change in protein phosphorylations
basically quite hinders a phosphoproteome analysis targeting human
postmortem brain samples. In fact, the present inventor and other
researchers have heretofore performed proteome-wide analyses, in
the postmortem human brain analysis, on the change in
phosphoproteins of mouse brains stored at room temperature or
4.degree. C. for different durations to determine a period during
which the brain would reflect the living state before death. As a
result, the present inventor and other researchers have revealed
that various phosphoproteins had already changed at a time point 12
hours after the preservation was started (Oka, T., Tagawa, K., Ito,
H. & Okazawa, H. "Dynamic changes of the phosphoproteome in
postmortem mouse brains," PLoS One, 2011, 6, e21405).
[0334] Hence, in view of this result, it was considered risky to
conduct a phosphoproteome analysis based solely on human samples.
Thus, efforts were made to identify phosphoproteins whose
expression amounts changed in Alzheimer's disease, by the following
stepwise approach: first, analyzing Alzheimer's disease model mice;
and then analyzing brain samples of Alzheimer's disease
patients.
[0335] To be more specific, first, the following five types of
transgenic mice (four types of AD model mice and one type of Tau
model mice) were dissected at the ages of 1, 3, and 6 months (4,
12, and 24 weeks old). Then, the cerebral cortex, hippocampus, and
striatum were quickly separated under a microscope and frozen
immediately. Note that all of these processes were completed within
5 minutes, as assessed by measuring the time with a stopwatch.
(1) PS1 transgenic mice (2) PS2 transgenic mice (3) Human
double-mutant APP695 transgenic mice (4) 5.times.FAD mice (5) Tau
transgenic mice.
[0336] Additionally, the background of the APP-Tg2576 mice and the
5.times.FAD mice was B6/SJL, the background of the PS1 transgenic
mice and the PS2 transgenic mice was C57BL6, and the background of
the Tau transgenic mice was C57BL6/C3H. Hence, these background
mice were utilized as control mice in the following experiment.
[0337] Note that, in the phosphoproteome analysis, preliminary
tests by AB SCIEX Triple TOF 5600 mass spectrometry were repeated
to determine the optimal conditions allowing the detection of the
largest number of phosphoproteins. Moreover, samples were
fractionated in multiple stages using cation exchange columns and
reverse-phase columns. Data obtained by combined analyses on the
same samples were merged. As a result, conditions (appropriate
amount, run time) for mass spectrometry allowing such quite a high
detection score of confidence 95% were obtained.
[0338] Next, using these conditions, a phosphoproteome analysis was
performed on the Alzheimer's disease model mice. To be more
specific, phosphoproteins were purified from the five types of
transgenic mice and the three types of background mice
corresponding thereto using TITANSPHERE.RTM. Phos-Tio Kit. Then,
after labeling with eight different probes using the iTRAQ reagent,
the analysis was performed by a single run of the mass
spectrometry. Subsequently, the systems biology analysis was
performed based on the experimental results of three mass
spectrometry analyses. As a result, with 95% confidence, 744 to 128
phosphoproteins were identified, and 13017 to 29995 phosphopeptides
were identified.
[0339] Thereafter, the proteins identified by nine mass
spectrometry analyses were mapped on the integrated protein-protein
interaction (PPI) database using a super computer. The utilized
integrated database was the genome network platform
(http://genomenetwork.nig.ac.jp/index_e.html) provided by National
Institute of Genetics. The integrated database includes the
experimentally-supported PPI database of the Human Genome Project
(GNP), BIND (http://www.bind.ca/), BioGrid
(http://www.thebiogrid.org/), HPRD (http://www.hprd.org/), IntAct
(http://www.ebi.ac.uk/intact/site/index.jsf), and MINT
(http://mint.bio.uniroma2.it/mint/Welcome.do).
[0340] After that, the mapped phosphoproteins were designated as
nodes. Further, proteins linked to significantly changed
phosphoproteins were attached as accessory nodes. Moreover, links
between the proteins were connected by lines (edges). Thus, a mouse
default network was prepared. Note that, in this network, proteins
indirectly linked to the identified proteins via two or more edges
were excluded from the network.
[0341] Next, although one protein had multiple P-values of multiple
peptides derived therefrom, these P-values were integrated by the
inverse normal method, and the integrated P-value was compared
between the model mice group and the control mouse group (n=3).
Then, as a result of the comparison between the AD model mice or
Tau model mice at the ages of 1 to 6 months and their control mice,
changed phosphoproteins were selected as nodes (p<0.05). Thus,
although unillustrated, networks of the phosphoproteins changed at
each time point of each model mouse were constructed.
[0342] Next, based on the constructed network of each model mouse
thus obtained, phosphorylation signal transductions commonly
changed in these model mice were identified using two different
approaches described below.
[0343] (1) The First Approach
[0344] This is a selection approach based on a result of a simple
comparison of phosphoproteome data from multiple model mice at the
same timepoint (hypothesis free approach)
[0345] (2) The Second Approach
[0346] This is a selection approach based on an assumption that an
abnormal phosphorylation signal is generated in some process of
amyloid aggregation or before the aggregation (A.beta.
aggregation-linked approach).
[0347] Note that, in the first approach, the number of common nodes
among the different models decreased with an increase in the number
of AD models compared. To be more specific, the number of common
nodes among the four types of AD models was only one (only MAP1B at
the age of 1 month), while no node was included at the ages of 3
and 6 months. Thus, for further analyses, although unillustrated,
65 nodes were selected which were common between two types of the
AD model mice one or more times. Among the 65 proteins, there were
51 proteins whose phosphorylations changed commonly in one
combination of two types of the AD model mice at a certain single
time point, while 14 proteins were phosphoproteins commonly changed
at multiple time points and phosphoproteins commonly changed in
multiple combinations of two types of the AD model mice (regarding
the 14 proteins, see FIG. 1).
[0348] Meanwhile, in the second approach, an immunohistological
analysis was performed on the four types of the AD model mice.
Then, pathological differences were confirmed among these model
mice. To be more specific, although unillustrated, in each of the
5.times.FAD mice and the APP mice, amyloid deposition started at
the ages of 3 and 6 months. On the other hand, in the PS1 mice and
the PS2 mice, no amyloid deposition was confirmed even at the age
of 6 months.
[0349] Thus, based on such a result, it was presumed that the
5.times.ADD mice and the APP mice should share the pathological
signal transduction at the time when the A.beta. deposition
started. Hence, significantly changed phosphoproteins were compared
between the 1-month-old 5.times.FAD mice and the 3-month-old APP
mice, or between the 3-month-old 5.times.FAD mice and the
6-month-old APP mice.
[0350] Then, from this comparison result, 11 nodes common in the
5.times.FAD mice and the APP mice were selected as phosphoproteins
deduced to be linked to A.beta. aggregation in the brain (regarding
the 11 nodes (proteins), see FIG. 1).
[0351] Surprisingly, as apparent from the result shown in FIG. 1,
collating the results obtained by the different approaches showed
that eight of the 11 proteins selected by the A.beta.
aggregation-linked approach were proteins also selected by the
hypothesis free approach. On the other hand, more than a half of
the 14 proteins whose phosphorylation states were observed to be
commonly changed at multiple time points between two types of the
AD model mice were also selected by the A.beta. aggregation-linked
approach.
[0352] Moreover, all of these phosphoproteins changed before the
onset. To be more specific, although unillustrated, the four types
of the AD model mice (PS1, PS2, APP, and 5.times.FAD) were
subjected to the behavioral tests (Morris water maze test, rotarod
test, open-field test, elevated plus maze test, light-dark box
test, and fear-conditioning test), but any abnormal behavior was
not detected at the age of 6 months.
[0353] As described above, in this phosphoproteomics analysis, the
two independent approaches arrived at the similar conclusion, and
17 phosphoproteins were identified as factors involved in a
pre-onset stage of Alzheimer's disease and composing a network (AD
core network) which played a central role in the pathology.
Example 2
[0354] <AD Core Network Analysis in Tau Model Animals>
[0355] Although no conclusion has been drawn yet regarding the
Alzheimer causative factor and onset mechanism, the most likely
mechanism is such that when amyloid .beta. molecules aggregate
(amyloid pathology), the aggregation promotes tau phosphorylation
and polymerization (tau pathology), consequently leading to nerve
cell death and so forth (amyloid cascade hypothesis). Hence,
regarding the transition from amyloid pathology to tau pathology
presumed in this hypothesis, the 17 proteins selected from the
amyloid-pathology-induced AD model mice were re-analyzed targeting
tau-pathology-induced AD model mice.
[0356] To be more specific, the 14 proteins also selected from the
AD model mice by the hypothesis free approach were compared with
phosphoproteins which changed in the Tau model mice. As a result,
it was revealed as shown in Table 1 that 10 phosphoproteins (ADDB,
NFH, NFL, SPTA2, BASP1, CLH, MARCS, NEUM, SRRM2, and Marcksl1) were
commonly changed between the AD model mice and the Tau model
mice.
[0357] Moreover, although unillustrated, tau was not included in
the 17 proteins but included in the 65 proteins detected by the
hypothesis free approach. To be more specific, the amount of the
phosphorylated tau protein was enhanced commonly in severe AD model
mice (5.times.FAD and APP) and tau model mice at the age of 1
month, supporting that the phosphorylation of the tau protein
linked amyloid pathology to tau pathology.
Example 3
[0358] <AD Core Network Analysis in Human AD Patients>
[0359] Next, the 17 proteins selected from the result of the AD
model mice described above were evaluated based on phosphoproteome
data on the brains of human AD patients.
[0360] To be more specific, first, in order to obtain
phosphoproteome data on the brains of human AD patients, mass
spectrometry was performed using five brains of human AD patients
as in the case of the mice. Note that the brain samples used in
this analysis were isolated, frozen, and stored within 1 hour after
death. The temporal lobe (temporal pole) and occipital lobe
(occipital pole) samples of the brains were subjected to the
analysis. Normally, these brain regions are remarkably affected
regions in AD patients. In addition, these brain samples were
analyzed after confirmed by a pathological examination that the
samples were not contaminated with tissues exhibiting no AD
pathology, such as lewy bodies and argyrophilic grains. Further,
five brains derived from healthy subjects matching with the AD
patients in age and five brains derived from patients having
dementia with lewy bodies were also used as controls in the
analysis.
[0361] Although unillustrated, based on phosphoproteins detected in
all the human brains as a result of the analysis, a human default
network was prepared. Note that, in this case, edges and accessory
nodes were added to nodes based on not only the human PPI database
but also mouse and rat PPI databases so as not to miss important
molecules such as kinases and phosphatases in comparing the human
and mouse networks in the subsequent analysis stage.
[0362] Then, each of the temporal lobe and occipital lobe was
compared between the AD patient brains and the normal brains or DLB
patient brains, and changed phosphoproteins were selected as "human
(AD)-(normal) nodes" or "human (AD)-(DLB) nodes". It was noteworthy
that all the disease-specific nodes in the "human (AD)-(DLB) nodes"
were also detected in the "human (AD)-(normal) node" network.
[0363] Subsequently, networks were prepared based on the "human
(AD)-(normal) nodes" in the temporal lobe and occipital lobe, and
compared with the above-described 17 proteins based on the mouse
phosphoproteome. The result revealed as shown in Table 1 that ADDB,
NFH, NFL, SPTA2, BASP1, G3P, MARCKS, and NEUM among the 17 proteins
composing the AD core network were changed commonly in the human AD
patients.
[0364] Moreover, these nine phosphoproteins commonly changed also
in the human AD patients were identified as the phosphoproteins
having been changed in the Tau model mice, except for G3P.
[0365] Thus, the above analysis results of the Tau model animals
and the human AD patients verified that the 17 proteins or at least
most of them were involved in a pre-onset stage of Alzheimer's
disease and were components of a network which played a central
role in the pathology.
Example 4
[0366] <Functional Analysis on AD Core Network>
[0367] Based on the integrated human-mouse PPI database described
above, the AD core network composed of the 17 proteins was
prepared. FIG. 2 shows the obtained result.
[0368] As apparent from the result shown in FIG. 2, surprisingly,
12 proteins in the 17 proteins were directly linked, and three
proteins (SRRM2, BASP1, and ADDB) were linked via one independent
protein. Note that, in the PPI database, Marcksl1 was a protein not
directly linked to the other 16 proteins but exhibiting a high
homology with MARCKS. This revealed that at least 15 proteins
formed a single functional network.
[0369] Further surprisingly, their functions were mainly directly
related to synapse functions such as spine formation, vesicle
recycling, and energy production.
[0370] SPTA2 (brain .alpha. spectrin) is a protein cross-liked to
actin and expressed at a high level in the brain (see Leto, T. L.
et al., Mol. Cell. Biol., 1988, Vol. 8, pp. 1 to 9). It is known
that SPTA2 interacts with SHANK at the postsynaptic density (see
Bockers, T. M. et al., J. Biol. Chem., 2001, Vol. 276, pp. 40104 to
40112), and interacts with ADDB/adducin-b, one of the 17 proteins
identified this time, forming spectrin/adducin/actin complexes (see
Li, X. et al., J. Biol. Chem., 1996, Vol. 271, pp. 15695 to 15702).
Moreover, it has also been revealed that these proteins are
substrates of protein kinase C (PKC), and after the phosphorylation
of these proteins, the complex becomes unstable, decreasing the
membrane stability, too.
[0371] It is known that ADDB contains a MARCKS-related domain, and
that the phosphorylation by PKC controls the postsynaptic
localization and inhibits actin/spectrin complex formation as
described above (see Matsuoka, Y. et al., J. Cell Biol., 1998, Vol.
142, pp. 485 to 497). Additionally, this system has been shown to
control synapse production and removal, although the result was at
the neuromuscular junction (NMJ) in Drosophila (see Pielage, J. et
al., Neuron, 2011, Vol. 69, pp. 1114 to 1131).
[0372] MARCKS, BASP1, and NEUM are known to be greatly involved in
signals originating from lipid rafts. Additionally, MARCKS is a
PKC-specific substrate and normally localized at the cell membrane.
However, it is known that after phosphorylated or bound to
calmodulin, MARCKS is released from the cell membrane and
transferred to the cytoplasm, inhibiting F-actin cross-linking (see
Hartwig, J. H. et al., Nature, 1992, Vol. 356, pp. 618 to 622).
Further, various morphological and functional abnormalities have
been observed in mouse brains having mutant MARCKS (see Stumpo, D.
J. et al., Proc. Natl. Acad. Sci. U.S.A, 1995, Vol. 92, pp. 944 to
948).
[0373] MRP/Marcksl1 is a member of the MARCKS family, and involved
in PKC signal transduction. Additionally, it is known that Marcksl1
is phosphorylated by JNK and controls the actin stability and the
filopodium formation of neurons (see Bjorkblom, B. et al., Mol.
Cell. Biol., 2012, Vol. 32, pp. 3513 to 3526).
[0374] NEUM/neuromodulin/GAP43 is an important component of growth
cone/axon presynaptic terminals and is known as a main substrate of
PKC (see Benowitz, L. I. et al., Trends Neurosci., 1997, Vol. 20,
pp. 84 to 91). Additionally, it is known that NEUM interacts with
various molecules. For example, there is a report that NEUM
interacts with PIP2 and palmitate, or cytoskeletal proteins such as
actin, spectrin, synaptophysin, and tau. As in the case of MARCKS
and SPTA2, it has been shown that NEUM is also controlled by
calmodulin and moves between the membrane and cytoplasm (see Gamby,
C. et al., J. Biol. Chem., 1996, Vol. 271, pp. 26698 to 26705). As
described above, NEUM is an adaptor protein which controls
presynaptic terminal functions via cytoskeletal regulation, and is
suggested to be involved in memory and LTP formation (see
Routtenberg, A. et al., Proc. Natl. Acad. Sci. U.S.A, 2000, Vol.
97, pp. 7657 to 7662).
[0375] BASP1/NAP-22/CAP23 is myristoylated protein having a PEST
motif, and is abundant in axonal terminals (see Mosevitsky, M. I.
et al., Biochimie, 1997, Vol. 79, pp. 373 to 384). Although the
function has not been sufficiently elucidated, BASP1/NAP-22/CAP23
exists at the inner surface of a lipid raft in the cell membrane.
In addition, BASP, MARCKS, and NEUM seem to regulate PI(4,5)P2 by a
common mechanism. Further, it is suggested that the
phosphorylation-dependent interaction between calmodulin and BASP,
MARCKS, or NEUM promotes actin network formation (see Laux, T. et
al., J. Cell Biol., 2000, Vol. 149, pp. 1455 to 1472).
[0376] As described above, it is suggested that the proteins
composing the AD core network form a network which controls
presynaptic and postsynaptoc morphologies.
[0377] Meanwhile, phosphoproteomic changes of SYT1/synaptotagmin 1
and GPRIN1/G protein regulated inducer of neurite outgrowth 1 were
not detected in the brains of both the Tau model mice and the human
AD patients. Nevertheless, these proteins might also be involved in
a pre-onset stage of AD. Particularly, SYT1/synaptotagmin 1 is
important because it controls vesicle recycling at synaptic
terminals. Note that SYT1 is known to form a complex at synaptic
terminals with a vesicle cargo molecule CLH/clathrin heavy chain
selected by both of the hypothesis free approach and the A.beta.
aggregation-linked approach (see Schwarz, T. L., Proc. Natl. Acad.
Sci. U.S.A, 2004, Vol. 101, pp. 16401 to 16402). Additionally,
GPRIN1 is a G.alpha.o effector enriched in the growth cone membrane
fraction which induces neurite outgrowth (see Chen, L. T., J. Biol.
Chem., 1999, Vol. 274, pp. 26931 to 26938). However, the detailed
functions have not been elucidated yet.
[0378] Further, interestingly, molecules involved in energy
production were also selected. To be more specific, ATPA and ATPB
are mitochondrial ATP synthase subunits A and B, respectively. ACON
is mitochondrial aconitase 2 which catalyzes citrate isomerization
in TCA cycle. Moreover, G3P/GAPDH/glyceraldehyde-3-phosphate
dehydrogenase is an important enzyme in glycolysis, also plays a
nuclear function through the nitrosylase activity, and affects
microtubule assembly by the same mechanism. Thus, the enzyme is
also related to cytoskeleton.
[0379] SRRM2 is involved in splicing together with SRm160 (see
Blencowe, B. J., Genes Dev., 1998, Vol. 12, pp. 996 to 1009), and
is functionally different from the other core network proteins.
[0380] HS90A/HSP90 is a chaperon molecule involved in quality
control and folding of various proteins. Because of the general
roles, HS90A/HSP90 is linked to various proteins in the core
network.
[0381] As described above, it was revealed that the
phosphoproteomic changes in the pre-onset stage of Alzheimer's
disease were selectively focused on two or three networks which
controlled synapse function and energy metabolism.
[0382] <Changes in Phosphoproteins Due to Aging in AD Model
Animals>
[0383] Further analyzed was how chronological changes in
phosphoproteins (changes in phosphoproteins by pathological aging)
in AD model mice were related to those (chronological changes in
phosphoproteins in the background mice) in normal aging
(physiological aging). Note that proteins from which data were not
obtained with a high confidence at any time point of the
5.times.FAD mice were excluded from this analysis.
[0384] In this analysis, three sets of new samples including the
5.times.FAD mice and the background mice at the ages of various
time points from 1 to 12 months were analyzed by the mass
spectrometry. Moreover, based on values of the 1-month-old
background mice obtained regarding the 17 phosphoproteins, values
of the 5.times.FAD mice and the background mice at the ages of each
month were corrected and plotted on a graph to analyze the
chronological changes in these phosphoproteins.
[0385] As a result, although unillustrated, the patterns of changes
in most of the phosphoproteins composing the AD core network were
qualitatively similar between the physiological aging and the
ageing due to the pathological aging. However, the patterns were
quantitatively different. It was revealed that each phosphoprotein
had a time point when a difference in the expression amount thereof
was remarkably large between the 5.times.FAD mice and the
background mice.
[0386] For example, regarding MARCKS, Marcksl1, and SRRM2, amounts
of these phosphoproteins in the 1-month-old 5.times.FAD mice were
remarkably large in comparison with those of the background mice.
Note that the difference was diminished over time. Moreover,
regarding SPTA2, G3P, ADDB, SYT1, BASP1, HSP90A, and NEUM, amounts
of these phosphoproteins in the 3-month-old 5.times.FAD mice were
remarkably large in comparison with those of the background mice.
Further, regarding NFH, NFL, CLH, and GPRIN1, amounts of these
phosphoproteins in the 12-month-old 5.times.FAD mice were
remarkably different from those of the background mice.
[0387] Furthermore, based on the result of analyzing the above
chronological changes in the phosphoproteins, the core protein
network was reconstructed. FIG. 3 shows the obtained result.
[0388] As described above, the phosphorylations of MARCKS and its
homolog Marcksl1 changed at the initial phase. Moreover, it was
revealed as shown in FIG. 3 that the changes of these were followed
by those of the other core phosphoproteins belonging to the same
functional domain or related functional domains.
Example 5
[0389] <Search for Kinases/Phosphatases Involved in Change in AD
Core Network>
[0390] As shown in FIG. 3, the chronological changes in the
phosphorylations of the proteins composing the AD core network in
the pre-onset stage of Alzheimer's disease can be categorized into
three patterns: one having a peak at the initial phase, one having
a peak at the mid phase, and one having a peak at the late phase.
Moreover, in view of these categories, it is presumed that the
phosphorylations of the proteins composing the AD core network are
controlled by particular kinases and the like in a time-specific
manner.
[0391] Hence, in order to search for kinases and the like involved
in this AD core network control, first, kinases and phosphatases
were selected among the proteins directly linked to the proteins
composing the AD core network. Then, among these kinases and
phosphatases, PKC was considered as the most important enzyme
involved in the change in the AD core network from the viewpoint
that it could phosphorylate the largest number of the core protein.
Note that, as having been already revealed from various reports in
the past regarding AD pathology, MAPK and MAPKKK were identified
after PKC.
[0392] Moreover, as the second group following these three kinases,
identified were casein kinase (CSKII), receptor-interacting protein
serine/threonine kinase 1/3 (RIP1/3), cyclin-dependent kinase 5/6
(CDK5/6), and protein kinase C-like protein 1 (PKN1). Further, as
the third group, identified were Ca2+/calmodulin-dependent kinase
(CaMKI/II), protein kinase D (PKD), and the like. Moreover,
Lck/Yes-related novel protein tyrosine kinase (Lyn) and other 20
kinases were identified as candidates to control the core network,
although these were linked to just one core protein.
[0393] FIG. 3 shows the enzyme-substrate relation obtained by
comparing the result of analyzing the above kinases linked to the
core phosphoproteins with the result of the chronological changes
in the core phosphoproteins in the 5.times.FAD mice described
above.
[0394] As apparent from the result shown in FIG. 3, the initial
phase (at the age of 1 month), the mid phase (at the age of 3
months), and the late phase (at the age of 6 months) patterns of
the core phosphoproteins were observed to have correlations with
PKC, Lyn, CamK, and CASK.
[0395] It seemed that the PKC family among these activated various
kinases earliest, and that the activation continued until the late
phase. To be more specific, it was revealed that first the PKC
family phosphorylated MARCKS and Marcksl1, and next the kinase
family phosphorylated G3P, NEUM, BASP1, and SPTA2.
[0396] Further, in order to identify target sequences of the
kinases which controlled 18 proteins composing the AD core network,
the data on the peptide phosphorylations obtained from the mass
spectrometry were examined again, and a phosphorylation level at
one site of each protein was individually analyzed. To be more
specific, identified were polypeptides containing one
phosphorylation site whose amount changed in comparison with the
wild type at P<0.05 at one or more time points in at least one
model among the five types of transgenic mice (the four types of AD
model mice and one type of Tau model mice).
[0397] As a result, although unillustrated, significant changes in
the phosphorylation levels were observed in ADDB at serine at
position 60, serine at position 62, serine at position 532, serine
at position 594, serine at position 602, serine at position 618,
serine at position 692, and serine at position 700. Significant
changes in the phosphorylation levels were observed in NFH at
serine at position 500, serine at position 535, serine at position
583, serine at position 673, serine at position 721, serine at
position 763, serine at position 795, serine at position 834,
threonine at position 839, serine at position 867, and serine at
position 888. Significant changes in the phosphorylation levels
were observed in NFL at serine at position 473, serine at position
523, and serine at position 532. Significant changes in the
phosphorylation levels were observed in SPTA2 at serine at position
1031 and serine at position 1217. Significant changes in the
phosphorylation levels were observed in ATPB at threonine at
position 262 and threonine at position 453. Significant changes in
the phosphorylation levels were observed in BASP1 at threonine at
position 31, threonine at position 36, serine at position 92,
serine at position 131, serine at position 192, and serine at
position 218. Significant changes in the phosphorylation levels
were observed in G3P at threonine at position 182 and threonine at
position 209. Significant changes in the phosphorylation levels
were observed in GPRIN1 at serine at position 182, serine at
position 219, serine at position 495, serine at position 576,
serine at position 691, serine at position 693, serine at position
714, serine at position 764, serine at position 771, serine at
position 816, and threonine at position 795. Significant changes in
the phosphorylation levels were observed in MARCKS at serine at
position 26, serine at position 27, serine at position 29, serine
at position 113, serine at position 122, serine at position 124,
serine at position 125, serine at position 127, serine at position
128, serine at position 138, serine at position 140, serine at
position 141, threonine at position 143, serine at position 163,
serine at position 171, and serine at position 299. Significant
changes in the phosphorylation levels were observed in NEUM at
serine at position 86, threonine at position 89, serine at position
96, serine at position 142, threonine at position 172, and serine
at position 193. Significant changes in the phosphorylation levels
were observed in SRRM2 at serine at position 1067, serine at
position 1278, serine at position 1305, serine at position 1339,
serine at position 1359, serine at position 1360, serine at
position 2351, serine at position 2084, serine at position 2404,
serine at position 2535, threonine at position 1448, and threonine
at position 2350. Significant changes in the phosphorylation levels
were observed in Marcksl1 at serine at position 22, threonine at
position 85, serine at position 104, threonine at position 148,
serine at position 189, serine at position 151, and serine at
position 185. Significant changes in the phosphorylation levels
were observed in HS90A at serine at position 231 and serine at
position 263. Significant changes in the phosphorylation levels
were observed in SYT1 at threonine at position 125 and threonine at
position 128.
[0398] <Involvement of Kinases in Alzheimer's Disease
Pathology>
[0399] The significance of the AD core network, which was
constructed based on the above-described analysis result, in the
pathology of Alzheimer's disease was verified using in vivo and in
vitro experimental systems.
[0400] The functions of the core factors revealed by the
phosphoproteome analysis suggested that specific phosphorylation
signals linking presynaptic cytoskeleton to postsynaptoc
cytoskeleton were perturbed at the earliest stage before the onset
of Alzheimer's disease. Particularly, it has been suggested that
the cytoskeleton network composed of actin binding proteins such as
actin and spectrin mainly controls dendritic spine formation (see
Matus, A., Science, 2000, Vol. 290, pp. 754 to 758, Tada, T. et
al., Curr. Opin. Neurobiol., 2006, Vol. 16, pp. 95 to 101). Hence,
it was presumed that the link from MARCKS to actin polymerization
and the link from SPTA2 to actin-spectrin cross-linking became
abnormal at the initial phase of Alzheimer's disease, and that
these cytoskeleton network activations affected the dendritic spine
dynamics, and were consequently involved in the pathology of
Alzheimer's disease.
[0401] Thus, the dendritic spine dynamics were analyzed with a
two-photon microscope targeting the 5.times.FAD mice (12 weeks old)
before the onset of Alzheimer's disease. FIGS. 4 to 9 show the
obtained result.
[0402] Living cortical neurons of layer 1 of the 12-week-old
5.times.FAD mice were observed with a two-photon microscope. The
result revealed as shown in FIGS. 4 and 5 that the dendritic spine
densities of the cortical neurons were remarkably decreased.
Moreover, decreases in the number of spines per dendritic shaft
length were observed in all the spine types (thin, mushroom, and
stubby) (see FIG. 6). Further, regarding absolute numbers of
dendritic spines per 100 .mu.m, all of the formed spines,
eliminated spines, and stably remaining spines were decreased (see
FIGS. 7 to 9). Moreover, regarding the percentages of three types
of spine dynamics in the 5.times.FAD mice, the formed spines were
decreased. On the other hand, no remarkable change was observed in
the eliminated spines and the stably remaining spines (see FIG. 9).
In sum, it was revealed that the spine formation in process was
affected in the pathology of the 5.times.FAD mice. Note that these
results basically agree with the findings in the past regarding AD
model mice (see Palop, J. J. et al., Neurosci., 2010, Vol. 13, pp.
812 to 818, Wei, W. et al., Nat. Neurosci., 2010, Vol. 13, pp. 190
to 196, Wu, H. Y. et al., J. Neurosci., 2010, Vol. 30, pp. 2636 to
2649).
[0403] Hence, next, kinases were analyzed which were suggested to
affect the AD core network from the initial phase to the mid phase
of the pre-onset stage of Alzheimer's disease. More concretely, the
effect of the kinases on the synaptic pathology in the 5.times.FAD
mice was analyzed by administering inhibitors and so forth against
these kinases into the subarachnoid spaces of the mice. FIGS. 4 to
9 show the obtained result.
[0404] Administering a PKC inhibitor (Go697) ameliorated spine
abnormality in the 5.times.FAD mice and increased the total spine
density (see FIGS. 4 and 5). Moreover, the numbers of spines of
thin, mushroom, and stubby types were also increased (see FIG. 6).
Further, regarding the spine dynamics, the Go697 treatment enhanced
the spine formation and stability (see FIGS. 7 to 9).
[0405] Moreover, it was revealed that the treatment with a CamKII
inhibitor (KN-93) also had a therapeutic effect on the spine
formation and stability. Further, it was also revealed that the
numbers of all the types of dendritic spines that had been
decreased in the 5.times.FAD mice were recovered by the treatment
(see FIGS. 7 to 9).
[0406] Further, it was revealed that the treatment with a Lyn
kinase activator (MLR1023) also had a therapeutic effect on the
spine formation and stability as in the case of the treatment with
the PKC inhibitor or CamKII inhibitor (see FIGS. 7 to 9). Note that
MLR1023 found to be effective this time is also known as an insulin
receptor potentiating agent. Hence, the therapeutic effect this
time is presumably also an effect of alleviating the insulin
resistance, which is observed in the brains of Alzheimer's disease
patient (see Craft, S., Nat. Rev. Neurol., 2012, Vol. 8, pp. 360 to
362).
[0407] Furthermore, to confirm the effects of the kinase inhibitors
in an in vivo system, an immunohistological analysis was performed
targeting brain samples after two-photon microscope observation,
and using antibodies against kinases in activated forms. FIGS. 10
and 11 show the obtained result.
[0408] As a result, it was verified as shown in FIGS. 10 and 11
that PKC.beta., PKC.delta., and CamKII (CamKII.alpha.) were
activated in the cortical areas (retrosplenial cortexes) of the
5.times.FAD mice. Moreover, the result of quantifying signals
generated from immunostained sites confirmed the above-described
effects of the kinase inhibitors.
[0409] <Involvement of MARCKS in Spine Pathology of Alzheimer's
Disease>
[0410] As described above, MARCKS was detected as a protein whose
phosphorylation changed at the initial phase of Alzheimer's disease
by the aging pattern analysis and also by the aggregation-linked
approach described above (see Table 1 and FIG. 3). This suggests
that MARCKS is the most reliable phosphorylation signal
transduction molecule in the pre-onset stage of the pathology of
Alzheimer's disease.
[0411] To confirm this, MARCKS, which is a substrate of PKC and
CamKII, was knocked down in AD model mice. To be more specific, a
lentiviral vector expressing a shRNA against MARCK was injected
into the cortexes of the 5.times.FAD mice (12 weeks old) before the
onset. FIGS. 12 to 18 show the obtained result.
[0412] As apparent from the result shown in FIGS. 12 to 18, the
spine pathology in the 5.times.FAD mice was ameliorated by
suppressing the expression of MARCKS with the shRNA. Moreover, the
shRNA against MARCKS recovered the decrease in the number of
spines, increased the numbers of immature and matured spines, and
further improved the spine formation in the 5.times.FAD mice.
Example 6
[0413] <Search for Kinase Involved in Transition from Amyloid
Pathology to Tau Pathology>
[0414] In addition to the identification of the specific network in
the pre-onset stage of Alzheimer's disease described above, efforts
were made to identify a kinase capable of promoting tau
phosphorylation.
[0415] The proteins selected based on the hypothesis free approach
described above did not directly include kinases/phosphatases, but
the enhancement of a b-raf protein amount was observed in two
3-month-old AD model mice (p<0.05). Moreover, the enhancement
was observed in three 1-month-old AD model mice and two 6-month-old
AD model mice, too.
[0416] Hence, whether or not inhibiting b-raf actually enabled
regulation of tau pathology was tested. To be more specific, an
amyloid .beta. protein (A.beta.1-42) and three types of b-raf
inhibitors were added to primary cultures of cortical nerve cells,
and tau phosphorylation in the cortical nerve cells was analyzed.
Note that it has been revealed that treating cortical nerve cells
with A3 enhances tau phosphorylation. FIGS. 19 and 20 show the
obtained result.
[0417] As apparent from the result shown in FIGS. 19 and 20, the
tau phosphorylation was suppressed in the A.beta.-treated cortical
nerve cells. Moreover, although unillustrated, the enhancement of
tau phosphorylation was commonly observed in severe AD model mice
(5.times.FAD, APP) and tau model mice at the age of 1 month. To be
more specific, this means the tau phosphorylation surprisingly
starts in the brains of young AD model mice (1 month old) before
immunohistological and symptomatic changes.
[0418] Thus, the above result suggested that b-raf was involved in
the promotion of the transition from amyloid pathology to tau
pathology.
[0419] --Frontotemporal Lobar Degeneration--
[0420] In the present Examples, next, analyses were performed by
employing experimental methods and so forth described below to
identify a signal transduction pathway which played a central role
in a pre-onset stage of frontotemporal lobar degeneration, and
consequently to provide target molecules useful in the diagnosis
and treatment of frontotemporal lobar degeneration.
[0421] Additionally, experiments other than those specifically
described below were conducted as in the case of the above
Alzheimer's disease analyses, unless otherwise specifically
stated.
[0422] <Frontotemporal Lobar Degeneration Model Mice>
[0423] In the present Examples, frontotemporal lobar degeneration
model mice were prepared to search for the target molecules.
[0424] It has been known that arginine at position 504 of the PGRN
(progranulin) protein is conserved across species, and that a point
mutation at this site mainly causes dementia (see Nicholson, A. M.
et al., Alzheimers Res. Ther. 4, 4 (2012), Le Ber, I. et al., Hum.
Mutat., 2007, Vol. 28, pp. 846 to 855).
[0425] Hence, in order to introduce a stop mutation into this site,
heterozygous PGRN-R504X mutation knockin mice were prepared. In the
preparation, a Neo cassette was inserted in C57BL/6J mice.
[0426] To be more specific, first, a targeting vector for the
knockin mice preparation was constructed using the following two
types of constructs.
[0427] (Construct 1)
[0428] A 6.5-kbp NotI-XhoI fragment with the R504X mutation was
amplified by PCR from a Bac clone (ID: RP23-311P1 or RP23-137J17)
and subcloned into a pBS-DTA vector (manufactured by Unitech Co.,
Ltd.).
[0429] (Construct 2)
[0430] A 2999-bp ClaI-XhoI fragment was amplified by PCR and
subcloned into a pBS-LNL(-) vector (manufactured by Unitech Co.,
Ltd.) with the Neo cassette.
[0431] Then, the BamHI (Blunt)-XhoI fragment derived from the
construct 2 was inserted between XhoI (Blunt)-SalI sites of the
construct 1 and subcloned. The vector thus obtained was used as a
targeting vector.
[0432] Next, the targeting vector was linearized by SwaI treatment,
and then introduced into ES clones of C57BL/6J mice by
electropolation. The genotype analysis of the ES clones was
performed by PCR. Positive clones in this analysis were analyzed by
Southern blotting using a probe for neomycin (Neo). Then, ES clones
confirmed to have homologous recombination occurred between the
targeting vector and the genome of the C57BL/6J mice were injected
into a mouse early embryo to prepare chimeric mice. Subsequently,
the chimeric mice were bred with CAC-Cre mice to remove the Neo
cassette in the F1 mouse genome. Further, using the genomic DNA
prepared from the tail of the F1 mouse thus obtained, individuals
in which the PGRN mutation was introduce by the knockin were
selected from these mice. Thus, PGRN-KI mice were established. Note
that, unless otherwise specifically stated, the PGRN-KI mice
described below refer to the heterozygotes.
[0433] Moreover, the PGRN-KI mice thus prepared were subjected to a
western blot analysis using an anti-PGRN antibody. It was confirmed
that an amount of the full-length PGRN protein expressed was
reduced in the cerebral cortexes of the mice. Further, as predicted
from the nonsense-mediated RNA decay mechanism, the quantitative
PCR confirmed that an amount of the mutant mRNA expressed was also
reduced in the PGRN-KI mice. Furthermore, PGRN of the PGRN-KI mice
was immunostained, and the result was collated with that of a
nerve-cell marker protein NeuN. From this, it was suggested that
the reductions of the PGRN in both the cerebral cortex and the
cerebellum were mainly attributable to the reduction in nerve
cells.
[0434] In addition, interestingly, the body weights of the PGRN-KI
mice at birth were lighter than those of mice having the same
genetic background used as a control (C57BL/6J, hereinafter also
referred to as "background mice"). Nonetheless, 20 weeks after
birth, the body weights of most of the PGRN-KI mice were not much
different from that of the wild type. Further, the weight of the
brain was slightly light in comparison with the control, but no
structural abnormality was observed.
[0435] <Recovery Experiment with Vemurafenib and shRNA>
[0436] A pharmacological recovery experiment was conducted on the
PGRN-KI mice as follows. To be more specific, an osmotic pump (1
.mu.l/hour, 1003D, manufactured by Durect Corporation) was
introduced into the subarachnoid cavity of a 12-week-old mouse, and
1.7 .mu.M vemurafenib (S1267, manufactured by Selleckchem
Chemicals) or PBS was supplied for 3 days. In addition, 0, 8, and
24 hours on Day 3 after the introduction, imaging was
performed.
[0437] Moreover, 3 .mu.l of a shRNA-Tau lentiviral vector
(sc-430402-V, manufactured by Santa Cruz Biotechnology Inc.,
1.times.10.sup.6 TU) or scrambled shRNA (SC-108080, manufactured by
Santa Cruz Biotechnology Inc., 1.times.10.sup.6 TU) was injected
into the same region as in the case of AAV1-EGFP (regarding
AAV1-EGFP, see <In Vivo Imaging with Two-Photon Microscope>
for Alzheimer's disease described above). In addition, 0, 8, and 24
hours on Day 5 after the shRNA injection, imaging was
performed.
[0438] <Analysis on Mislocalization of Tau Protein in
Spine>
[0439] A coimmunostaining analysis was performed using an
anti-PSD-95 antibody and an anti-phosphorylated tau antibody
(Ser203 or Thr220). To be more specific, paraffin sections (5
.mu.m) were prepared from retrosplenial cortex (RSD) tissues of the
PGRN-KI mice and so on, co-stained with the antibodies, and
observed by LSM510 confocal microscope (manufactured by Zeiss,
objective magnification: .times.63, zoom 1, Z-stack images were set
at intervals of 0.8 .mu.m).
[0440] In the co-localization analysis, the number of sites where
phosphorylated tau and PSD-95 signals over lapped with each other
was counted in the obtained images (143 .mu.m.times.143 .mu.m).
[0441] Moreover, the fluorescent signal intensity derived from the
phosphorylated tau or PSD-95 was quantified using ZEN lite 2012
(manufactured by Zeiss). An average pixel intensity per ROI (20
.mu.m.times.20 .mu.m) was calculated.
[0442] Then, data were obtained from randomly set 10 images, and
used for the comparison between the mouse groups by statistical
analyses (one-way analysis of variance and Tukey's multiple
comparison test).
Reference Example
[0443] <Phenotype Analysis on PGRN-KI Mice>
[0444] First, analyzed was whether or not the PGRN-KI mice having
the stop mutation introduced in the PGRN (progranulin) gene as
described above would exhibit the frontotemporal lobar degeneration
(FTLD) phenotype.
[0445] PGRN-related FTLD is classified as FTLD-TDP characteristized
by TDP43 aggregates in the nucleus and cytoplasm. Note that TDP43
is a nuclear protein involved in RNA processing, but the aggregate
may be formed by the cytoplasmic translocation.
[0446] Hence, brain tissues of the PGRN-KI mice were stained with
an anti-TDP43 antibody. As a result, although unillustrated, signal
intensities of the TDP43 staining were not uniform in the frontal
cortexes of the PGRN-KI mice from the age of 1 month. Moreover, the
number of nerve cells not stained or weakly stained with the
anti-TDP43 antibody was apparently increased in the PGRN-KI mice in
comparison with that of the background mice. Further, the
difference became remarkable over time.
[0447] In addition, cytoplasmic inclusion, lentiform intranuclear
inclusion, and cytoplasmic TDP43 staining were observed in the
PGRN-KI mice. Further, in the mice, ubiquitin-positive aggregates
were also observed.
[0448] These characteristics were pathological findings observed in
PGRN-related FTLD of human. Thus, it was revealed that the PGRN-KI
mice were pathologically similar to patients of this disease.
[0449] On the other hand, p62 and FUS inclusion, which are rarely
observed in human pathology, were observed in the PGRN-KI mice as
an a typical finding. These inclusions recognized by an anti-p62
antibody or an anti-FUS antibody were detected in the frontal
cortexes (M2) at the age of 4 months, and spread to the parietal
cortexes at the age of 6 months. Meanwhile, in the PGRN-KI mice, an
apparent increase in apoptosis, which would be detected by Tunel
staining, was not observed until the age of 12 months.
[0450] Further, in addition to the analysis on the protein
aggregate, whether or not an inflammation was activated in the
PGRN-KI mice was analyzed. Note that, such inflammation activation
is a characteristic commonly observed across many neurodegenerative
diseases. An immunostaining was performed on the PGRN-KI mice using
an anti-IBA1 antibody and an anti-GFAP antibody. The result
revealed that inflammation was activated in each of microglia and
astrocytes. Moreover, such inflammation activations were also
confirmed by quantitative PCR targeting IL-1b and Cox-2. However,
the invasion of CD4- or CD8-positive cells was not observed.
[0451] Further, another common characteristic of neurodegenerative
diseases includes DNA damage. An apparent increase in .gamma.H2AX
focus formation was observed in cortical nerve cells at the age of
6 months. This revealed that DNA damage occurred in the PGRN-KI
mice, too.
[0452] Furthermore, the PGRN-KI mice were also subjected to six
behavioral tests as in the case of Alzheimer's disease model mice
described above. As a result, abnormalities regarding anxiety
memory and anxiety-related memory were not observed in the
open-field test, the light-dark box test, and the elevated plus
maze test. Nevertheless, an apparent decrease in memory formation
was observed in the fear-conditioning test. Note that it can be
said that this decrease in the memory formation was not due to a
disorder in a sensory function or motor function because no
abnormal score was observed in the rotarod test. Moreover, in the
Morris water maze test, a statistically significant decrease was
observed from the age of 3 months in the time during which the mice
stayed in the target region or the number of times the mice passed
through the target. This result supported the loss of the memory
formation in the PGRN-KI mice. Note that, in the Morris water maze
test using the PGRN-KI mice, the mice received the 60-second trial
four times a day for 5 days to learn the position of the platform
(target region). Then, the test was conducted under a condition
where the platform was removed to measure the time during which the
mice stayed in the target region where the platform was originally
located and the number of times the mice passed through the target.
Additionally, these characteristics observed in the behavioral
tests basically agree with clinical symptoms of FTLD patients
having an R504 stop mutation which mainly develops dementia.
[0453] As described above, the PGRN-KI mice reflected both the
pathological observations and the clinical symptoms of FTLD
patients, revealing that the mice were quite useful as FTLD model
animals.
Example 7
[0454] <Phosphoproteome Analysis on FTLD>
[0455] Using the PGRN-KI mice whose usefulness as FTLD model
animals was verified, efforts were made, as in the case of the
above Alzheimer's disease analyses, to comprehensively analyze
(phosphoproteome analysis) phosphorylation signal transductions in
FTLD also to identify a phosphorylation signal transduction which
played a central role in a pathology of the disease.
[0456] Particularly, PGRN has been reported to exhibit an
antagonistic action against TNF; on the other hand, contradictory
results have also been reported (see NPLs 17 to 21). To elucidate
this contradiction, a comprehensive proteome analysis was performed
on the cerebral cortex tissues of the PGRN-KI mice to examine, in
the brains of the PGRN mutation-related FTLD model mice, whether a
TNF signal transduction pathway was activated or a different type
of signal transduction pathway was activated.
[0457] To be more specific, the comprehensive proteome analysis was
performed as in the case of Alzheimer's disease described above
using ABSCIEX 5600 and targeting the cerebral cortex tissues
derived from three PGRN-KI mice and those derived from three
background mice (C57BL/6J).
[0458] Then, based on the comprehensive proteome data thus
constructed, whether or not the TNF signal transduction pathway was
activated in the cerebral cortex tissues of the PGRN-KI mice was
examined. Concretely, using signal transduction pathway-related
database (http://www.genome.jp/kegg/pathway.html) of KEGG (Kyoto
Encyclopedia of Genes and Genomes), proteins belonging to the TNF
signal transduction pathway were searched for proteins whose
phosphorylation states changed in the PGRN-KI mice and C57BL/6J
mice. As a result, surprisingly, no protein whose phosphorylation
changed was found in the TNF signal transduction pathway per se for
1 to 6 months after birth and after the onset.
[0459] Hence, next, an analysis was performed targeting 16
TNF-related signal transduction pathways including an adipocytokine
signal transduction pathway, a NF-kB signal transduction pathway,
and an apoptosis signal transduction pathway. The result revealed
that, in a MAPK signal transduction pathway, an mTOR signal
transduction pathway, and a signal transduction pathway related to
antigen processing and presentation, phosphorylations of proteins
belonging to these signal transduction pathways remarkably changed
in the PGRN-KI mice in comparison with the C57BL/6J mice.
[0460] Further, it was also revealed that, in these signal
transduction pathways, the most remarkable change in the
phosphorylation was focused on the MAPK signal transduction pathway
which would lead to tau protein phosphorylation.
[0461] Note that the mTOR signal transduction pathway and the MAPK
signal transduction pathway were common in PKC activation. On the
other hand, in the signal transduction pathway related to antigen
processing and presentation, the protein phosphorylation varied for
1 to 6 months after birth.
[0462] The MAPK signal transduction pathway was apparently
activated in the PGRN-KI mice from the pre-onset stage. During the
period of symptom progression also, multiple proteins belonging to
the signal transduction pathway were in high phosphorylation states
all the time.
[0463] Particularly, in seven proteins, b-raf, PKC.alpha.,
PKC.beta., PKC.gamma., tau, MAP2K1 (mitogen-activated protein
kinase kinase 1, MAP kinase kinase 1, MEK-1), and stathmin
belonging to the MAPK signal transduction pathway, the
phosphorylations at one or two amino acid sites of each of these
proteins (phosphopeptide amounts detected by the mass spectrometry)
remarkably changed in the PGRN-KI mice in comparison with the
C57BL/6J mice.
[0464] Importantly, the phosphorylation of the tau protein
significantly changed at multiple sites. Particularly, serine at
position 203, threonine at position 220, and serine at position 393
of the tau protein (corresponding respectively to serine at
position 214, threonine at position 231, and serine at position 404
of human tau protein) were in high phosphorylation states all the
time during the above-described period, or the phosphorylations
were enhanced over time.
[0465] Moreover, regarding b-raf and PKC.gamma. also, one or
multiple sites thereof were in high phosphorylation states all the
time, or the phosphorylations were enhanced over time.
[0466] More concretely, in the PGRN-KI mice, the phosphorylation of
serine at position 348 of b-raf was 1.1487 times, 1.1795 times, and
1.3664 times (shown are relative values at the ages of 1 month, 3
months, and 6 months, respectively) as high as those of the
C57BL/6J mice. Moreover, regarding serine at position 766 of b-raf,
the phosphorylation was 1.7508 times and 3.0476 times (shown are
relative values at the ages of 3 months and 6 months,
respectively). Further, regarding serine at position 769 of b-raf,
the phosphorylation was 1.9752 times (shown is a relative value at
the age of 6 months). Note that these serine at position 348,
serine at position 766, and serine at position 769 of b-raf
correspond respectively to serine at position 365, serine at
position 729, and serine at position 732 in human.
[0467] In addition, for PKC.gamma., in the PGRN-KI mice, the
phosphorylation of threonine at position 655 was 1.2052 times,
1.1308 times, and 1.5702 times (shown are relative values at the
ages of 1 month, 3 months, and 6 months, respectively) as high as
those of the C57BL/6J mice. Moreover, regarding serine at position
690 of PKC.gamma., the phosphorylation was 2.5918 times (shown is a
relative value at the age of 1 month). Note that these threonine at
position 655 and serine at position 690 of PKC.gamma. correspond
respectively to threonine at position 655 and serine at position
690 in human.
[0468] Moreover, although unillustrated, regarding serine at
position 766 of b-raf and threonine at position 655 of PKC.gamma.,
it was confirmed by western blotting that the phosphorylations at
these sites in the cerebral cortexes of the 3-month-old PGRN-KI
mice were enhanced in comparison with those in the 3-month-old
C57BL/6J mice as in the above result of mass spectrometry.
[0469] Further, the phosphorylation of MEK-1 (Map2k1) located
downstream of b-raf and PKC.gamma. was also analyzed by western
blotting. It was confirmed that the phosphorylation in the cerebral
cortexes of the PGRN-KI mice at the age of 3 months was also
enhanced in comparison with that of the 3-month-old C57BL/6J
mice.
[0470] Furthermore, an immunohistological analysis using an
anti-phosphorylated tau antibody AT8 was performed to detect
phosphorylated tau protein in the cytoplasms of the frontal lobe
nerve cells of the PGRN-KI mice. As a result, a signal of the
phosphorylated tau protein was detected at the age of 12
months.
[0471] On the other hand, a signal transduction induced by TNF was
analyzed by a co-immunoprecipitation method based on amounts of
complexes formed in the signal transduction (TNFR-TRADD complex,
TNFR-RIP complex, and TNFR-TRAF2 complex). As a result, no
significant difference was found between the PGRN-KI mice and the
C57BL/6J mice.
[0472] The above results revealed that, in the PGRN-KI mice, the
MAPK signal transduction pathway was activated, while the TNF
signal transduction pathway was not activated.
Example 8
[0473] <Analysis on Therapeutic Effect of b-Raf Inhibitor on
Behavioral Phenotype of FTLD Model Mice>
[0474] Analyzed was whether or not suppressing an abnormal
activation in the MAPK signal transduction pathway by using a b-raf
specific inhibitor would recover the behavioral phenotype of the
PGRN-KI mice.
[0475] To be more specific, in accordance with the protocol shown
in FIG. 21, a b-raf specific inhibitor vemurafenib or PBS was
provided to the 6-week-old PGRN-KI mice every day over 6 weeks.
Then, the behaviors of these mice were evaluated in the Morris
water maze test and the fear-conditioning test. FIGS. 22 and 23
show the obtained result.
[0476] As apparent from the result shown in FIGS. 23 and 23,
administering vemurafenib to the PGRN-KI mice remarkably recovered
the scores in the two tests.
[0477] Meanwhile, the therapeutic effect of thalidomide was also
tested. Note that thalidomide is known to suppress the TNF signal
transduction pathway. Concretely, in accordance with the protocol
shown in FIG. 24, thalidomide was administered to the PGRN-KI mice
by peritoneal cavity injection every day for the same period as
that in the protocol using the b-raf inhibitor. The behaviors of
these mice were evaluated by the above two tests. FIGS. 25 and 26
show the obtained result.
[0478] As apparent from the result shown in FIGS. 25 and 26, in the
thalidomide administration example also, beneficial effects were
finally verified regarding the scores in the Morris water maze test
and the fear-conditioning test.
[0479] Note that such symptom recoveries were not observed in the
PBS-administered PGRN-KI mice.
[0480] Moreover, the cerebral cortexes of the PGRN-KI mice treated
with the agent were analyzed by western blot. The result confirmed
as shown in FIG. 27 that vemurafenib suppressed the b-raf
phosphorylation. Further, it was revealed that vemurafenib also
suppressed the PKC phosphorylation.
[0481] On the other hand, in the thalidomide administration
example, the b-raf phosphorylation was suppressing, but the PKC
phosphorylation was not suppressed, as apparent from the result
shown in FIG. 28.
[0482] In sum, these results revealed that the two agents had the
therapeutic effects on the FTLD-involved phenotype through the
inhibition of the b-raf pathway.
Example 9
[0483] <Analysis on Recovery Effect of b-Raf Inhibitor and Tau
Knockdown on Spine Phenotype of FTLD Model Mice>
[0484] It is known that, in Alzheimer's disease and FTLD-Tau, tau
phosphorylation is involved in formation of paired helical
filaments (PHF) and aggregation of this protein in the cytoplasm.
Moreover, there is a report on a pathological relation between tau
and amyloid .beta. (A.beta.) in Alzheimer's disease. From these
findings, tau is normally believed to be an effector molecule
located downstream of A.beta..
[0485] In addition, it has recently been revealed that, in an
initial pathological stage when A.beta. exhibits toxicity on
synapse function, tau plays a different important role in synaptic
spines.
[0486] Further, it is also reported that the transition of tau to
spine due to a Fyn kinase which phosphorylates NMDAR enhances a
calcium concentration and triggers the destruction of spine
cytoskeleton.
[0487] Hence, based on the above findings, two hypotheses were
proposed and examined. To be more specific, as the first
hypothesis, the aforementioned therapeutic effect by suppressing
b-raf phosphorylation was presumably based on the elimination of
protein aggregation in the nerve by vemurafenib or thalidomide.
Accordingly, analyzed was the recovery effect of vemurafenib or
thalidomide on the protein aggregation in the cerebral cortexes of
the PGRN-KI mice.
[0488] However, although unillustrated, in an immunohistological
analysis on the brains of the PGRN-KI mice, no significant effect
of vemurafenib or thalidomide was observed on FUS and p62 inclusion
(IB). Moreover, regarding the transition of TDP43 into the
cytoplasm also, no large change was observed in the PGRN-KI mice in
which vemurafenib or thalidomide was administered.
[0489] Hence, next, the second hypothesis was proposed that
synaptic spines were impaired by abnormal tau phosphorylation. To
be more specific, the therapeutic effect of vemurafenib or
thalidomide was presumably exhibited through the recovery of
synaptic spines by suppressing b-raf phosphorylation, and in vivo
imaging was performed on synaptic spines with a two-photon
microscope.
[0490] Concretely, EGFP-expressing AAV was injected into
retrosplenial cortexes (RSD) of the PGRN-KI mice and the C57BL/6J
mice. Then, two weeks thereafter, in vivo imaging was performed on
synaptic spines with a two-photon microscope. FIGS. 29 and 30 show
the obtained result.
[0491] As shown in FIG. 29, the result of the in vivo imaging on
EGFP-positive nerve cells in layer 1 revealed that the spine
density was remarkably reduced in the PGRN-KI mice. Note that the
other spine parameters such as length, diameter, and volume were
not much different from those of the C57BL/6J mice. These suggest
that the reduction in the spine density is a phenotype requiring a
comparatively long time.
[0492] Further, as shown in FIG. 30, the result of observing the
spine dynamics at three time points showed that, consistently, the
numbers of produced spines, eliminated spines, and stably remaining
spines did not significantly change at any time point within 24
hours.
[0493] In addition, analyzed was whether or not vemurafenib and/or
tau knockdown enabled recovery of spine related phenotype. FIGS. 31
and 32 show the obtained result.
[0494] As shown in FIG. 31, the observation result with a
two-photon microscope revealed that administering vemurafenib using
an osmotic pump recovered the number of spines of the PGRN-KI
mice.
[0495] Moreover, as shown in FIG. 32, the in vivo recovery effect
on the number of spines was observed as a result of knocking down
Tau using a lentiviral vector expressing shRNA against tau
(sh-tau).
[0496] As shown in FIGS. 31 and 32, the changes in synaptic spines
were the same in the two treatments, but a slight difference was
detected in the static spine morphology. To be more specific, a
trend was observed that b-raf inhibition decreased the spine
volume. On the other hand, the tau knockdown tended to increase the
spine volume. This difference between the trends was conceivably
because the spines increased by vemurafenib and sh-tau were thin
spines in the former, but were thick spines in the latter.
Nevertheless, as shown in FIGS. 31 and 32, the difference was not
stably confirmed as a statistically significant difference.
[0497] In addition, the spine dynamics were also observed. However,
as shown in FIG. 33, no change was detected in spine production or
elimination.
[0498] The above results revealed that the vemurafenib
administration or tau knockdown had a recovery effect on the number
of spines. Moreover, these results confirmed that activating the
MAPK pathway including tau was a major mechanism of the abnormal
behavior in the PGRN-KI mice.
INDUSTRIAL APPLICABILITY
[0499] As has been described above, it has been revealed that, in
the pre-onset stage of Alzheimer's disease, stepwise enhancement of
the phosphorylation of the AD core network composed of MARCKS,
Marcksl1, SRRM2, SPTA2, ADDB, NEUM, BASP1, SYT1, G3P, HS90A, CLH,
NFH, NFL, GPRIN1, ACON, ATPA, and ATPB affects dendritic spine
dynamics and the like, consequently developing Alzheimer's disease.
Moreover, it has also been revealed that the phosphorylation of the
AD core network is caused by PKC, CaMK, CSK, and Lyn, and further
that b-raf is involved in the promotion of the transition from
amyloid pathology to tau pathology (enhancement of the
phosphorylation of the tau protein) important for the progression
of Alzheimer's disease.
[0500] Thus, the present invention targets the proteins composing
the AD core network and kinases which phosphorylate these proteins,
and is useful in providing early-stage diagnosis and treatment
methods against Alzheimer's disease and agents utilizable in these
methods.
[0501] In addition, regarding frontotemporal lobar degeneration
(FTLD) also, it has been revealed that TNF-related signal
transduction pathways, particularly a MAPK signal transduction
pathway, are activated from a pre-onset stage of the disease, and
that the activation decreases the number of synaptic spines in FTLD
patients, consequently developing abnormal behaviors and the
like.
[0502] Thus, the present invention targets b-RAF belonging to the
MAPK signal transduction pathway, and is useful in providing
diagnosis and treatment methods against FTLD and agents utilizable
in these methods.
* * * * *
References